Hindawi Publishing Corporation
Journal of Biomedicine and Biotechnology
Volume 2011, Article ID 830573, 9 pages
TheEvolution of theMitochondria-to-Calcium ReleaseUnits
1Department of Cell and Developmental Biology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
2Center for Research on Ageing and Department of Neuroscience and Imaging, Universit` a Gabriele d’Annunzio,
66100 Chieti, Italy
Correspondence should be addressed to Clara Franzini-Armstrong, email@example.com
Received 5 July 2011; Accepted 10 August 2011
Academic Editor: Aikaterini Kontrogianni-Konstantopoulos
Copyright © 2011 C. Franzini-Armstrong and S. Boncompagni. This is an open access article distributed under the Creative
Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the
original work is properly cited.
The spatial relationship between mitochondria and the membrane systems, more specifically the calcium release units (CRUs)
of skeletal muscle, is of profound functional significance. CRUs are the sites at which Ca2+is released from the sarcoplasmic
reticulumduringmuscle activation. Closemitochondrion-CRUproximity allows theorganelles totake upCa2+andthus stimulate
aerobic metabolism. Skeletal muscles of most mammals display an extensive, developmentally regulated, close mitochondrion-
CRU association, fostered by tethering links between the organelles. A comparative look at the vertebrate subphylum however
shows that this specific association is only present in the higher vertebrates (mammals). Muscles in all other vertebrates, even if
capable of fast activity, rely on a less precise and more limited mitochondrion-CRUproximity, despite some tethering connections.
This is most evident in fish muscles. Clustering of free subsarcolemmal mitochondria in proximity of capillaries is also more
frequently achieved in mammalian than in other vertebrates.
Mitochondria have two functional requirements: they need
oxygen and also some stimulation by Ca2+[1–3]. The latter
event has been disputed for a while, but it is now clear that
mitochondria take up some of the Ca2+released from the
endoplasmic reticulum (ER) under physiological conditions,
a step that has important effects on a variety of their func-
tions, including the stimulation of aerobic metabolism. The
uptake has been initially difficult to pinpoint, but specifically
targeted aequorin  gave a first clear evidence for Ca2+
entry into the mitochondria of living cells, and fluorometric
measurements of mitochondrial dehydrogenases activity
provided an early fast measurement of mitochondrial Ca2+
uptake in vivo . The concept has been developed that this
uptake is strictly dependent on a planned proximity between
the organelles and certain components of the ER. The so-
called high Ca2+microdomain hypothesis proposes that the
proximity is necessary because the relatively low mitochon-
drial affinity for Ca2+[1, 6–8] means that uptake will occur
only if the mitochondrion is near the source, so that it expe-
riences a sudden increase of the cytoplasmic Ca2+level to
relatively high values [9–11]. Close proximities between ER
and mitochondria are indeed frequent and well documented
in liver cells, where the ER, both RER and SER, is frequently
tightly wrapped around the mitochondria profiles [12, 13]
even in organisms as low as fish. Visible tethering connec-
tions between ER and mitochondria are sufficiently strong in
liver and myocardium of mammals to maintain the associ-
ation during cellular fractionation [14, 15]. More recently,
an even more direct interaction between ER components
and the outer mitochondrial membrane has been proposed,
based on a physical link between the voltage-dependent
anion channel (VDAC) of the outer mitochondrial mem-
brane and Ip3 receptors of the ER, allowing direct coupling
between Ca2+release and mitochondrial uptake .
Cardiac and skeletal muscle cells are inherently crowded
due to their content of myofibrils, and the mitochondria
share the narrow intermyofibrillar spaces with the sarcoplas-
mic reticulum (SR); transverse (T) tubules; dyads/triads;
2 Journal of Biomedicine and Biotechnology
glycogen granules and lipid droplets as well as the basic
cytoskeletal network. Among these organelles, the tri-
ads/dyads, constituted of junctional SR (jSR) and T tubules,
exits the SR during muscle activation. CRUs are located at
around the myofibrils following the path of the T tubule
network. Given the narrowness of the shared spaces, it is not
surprising that mitochondria and CRUs often rub elbows.
In skeletal and cardiac muscles of rodents, the proximity
is sufficient for mitochondria to take up Ca2+even during
the time course of a single twitch [17, 18]. Even though the
uptake by individual organelles is small, it can affect the rate
of relaxation in fibers that are rich in mitochondria  and
in rat fibers the buffering action of mitochondria may be
sufficient to reduce the frequency of detectable spontaneous
Pertinent questions are (1) is the CRU-mitochondria
relationship random, or is there a higher structural hierarchy
that specifically establishes the relative positioning of the two
organelles in skeletal muscle and (2) is the same structural
relationship present in muscles of all vertebrates. Structural
and functionalevidencepoints to a planned 3-D relationship
between the two organelles [21, 22] but not for all mito-
chondria and perhaps not in all muscles. Taking advantage
of different kinetic properties of Ca2+chelators, Shkryl and
Shirokova  demonstrated that two functional categories
of mitochondria coexist in skeletal muscle of the rat: those
that take up some of the Ca2+released by the SR during
muscle activation even in the presence of fast Ca2+chelators
and those that do not. The former are, it must be presumed,
closely juxtaposed to CRUs, the latter are at some distance.
The structural equivalent of this functional phenomenon
is the well-specified position of mitochondria relative to
CRUs in the muscle fibers from the same species, as well
as from other mammals. Muscles of course are of primary
importance in any activity from food gathering, to eating,
to defense and, indirectly, to reproduction and in some
cases to heat production. Based on the fundamental tenet
of evolution that features offering survival advantage are
retained, it may be expected that any variation essential for
the effective performance of muscles would appear as an
early event in evolution. As an example, the orderly arrange-
offering the possibility of rapid movements, is an early
evolutionary event, that is found in insects, all chordates,
and, even, in a slightly rudimentary form in some molluscan
muscles. An early appearance of a tight mitochondrion/CRU
relationship in vertebrate evolution would indicate that
this particular arrangement is of overriding functional
usefulness to both mitochondria and skeletal muscle; a later
appearance would indicate that it is advantageous but not
In order to establish the evolutionary significance of
specific mitochondria/CRU relationships, we launched a
widespread exploration of muscle ultrastructure within the
vertebrate subphylum. To that effect, we examined muscle
samples from a few organisms in each class, from fish to
mammals, relying on the existing literature for further data.
Skeletal muscles were fixed in a variety of vertebrates.
Most images came from an extensive archive present in the
laboratory, other from muscles that were fixed specifically
for this project. Fish: Lampetra planeri (larvae); Eptatretus
stouti (Hagfish); Lepisosteus osseus (Gar fish); Citharichthys
sordidus (pacific sand dab); Danio rerio (zebrafish); Poecilia
reticulata (guppy); Poecilia latipinna var. (black molly),
toadfish (Opsanus tau); amphibia (Rana pipiens, R. tem-
poraria); Reptiles (Boa constrictor, Nerodia sipedon, Anolis
carolinensis); Birds (Meleagris gallopavo; fringilla sp., Gallus
gallus); Mammals (Mus rattus, Rattus sp., Felis catus).
The animals were euthanized by a variety of means
(cervical dislocation and/or an overdose of anesthetic: CO2,
ether, isoflurane, sodium pentobarbital). After euthanasia,
the muscles were exposed and either fixed in situ by dripping
the fixative on them, or carefully dissected tendon-to-
tendon, pinned in Sylgard dish (Dow Corning) at resting
length and immersed in fixative. Fixation was in 3–9% glu-
perature. The muscles were stored in fixative at 4◦C for vari-
able periods of time, then postfixed in 2% OsO4in the same
buffer for 1-2hr at 4◦C, en block stained in saturated uranyl
acetate, with several washes after each step, and embedded
in Epon 812. Muscles for domestic chicken and kangaroo
(Macropus sp.) were simply obtained from the supermarket,
and small samples were treated as the freshly dissected mus-
crotome Leica Ultracut R (Leica Microsystem, Austria) using
a Diatome diamond knife (Diatome Ltd. Biel, Switzerland)
and stained in uranyl acetate and lead citrate solutions.
3.Results and Discussion
3.1. Mammals. The following description, based on pub-
lished electron micrographs of various muscles from labora-
tory rat and mouse [22, 24–26] and extensive unpublished
observations by the two authors, offers the background
for the overall distribution of mitochondria in muscles of
placental mammals. Many mitochondria in these muscles
lie within transverse planes that are positioned in close
Z line, and thus have a preferential location opposite to the
I bands of the sarcomere (Figure 1(a)). These mitochondria
are very thin and elongated and closely follow the junctional
SR sacs of CRUs over long distances, frequently coming
to distances of ∼25nm from the SR surface (Figure 1(b);
. Some fibers (e.g., type IIX and IIB in mouse) have
almost exclusively this type of mitochondrial disposition. A
second additional set of mitochondria is present in fibers
that are richer in these organelles (e.g., type I and IIA in
mouse). These mitochondria are larger in diameter; they
are longitudinally oriented and may span the distance of
several sarcomeres, encompassing I and A bands and Z
lines. Appropriate sections show that these mitochondria
extend transversely oriented arm-like branches as they pass
at the level of the I band and these branches are those that
follow along the length of triads (Figure 1(b)). It is not clear
Journal of Biomedicine and Biotechnology3
Figure 1: Cross (a) and longitudinal (b) sections of muscle fibers
from the mouse EDL muscle. In (a), slender, elongated approxi-
mately cylindrical mitochondria profiles occupy the intermyofib-
rillar spaces, running transversely for long distances between the
runs from left to right at a slight angle. Longitudinally arranged
that correspond to those seen in (a). The transverse mitochondria
branches run parallel to triads (CRUs) and are closely opposed to
the junctional SR (arrows) over long distances.
whether all the longitudinally arranged mitochondria are
directly connected to transverse extensions, but in general
it can be assumed that these two sets of mitochondria are
part of the same continuum. A different set of mitochondria
(not shown) resides at peripheral sites, between the most
peripheral myofibrils and the plasmalemma. These mito-
chondria are piled up into irregular mounds that project
over the fiber surface and they are closely apposed to each
other, but at some distance from the nearest myofibrils
and CRUs. Capillaries are usually located in proximity of
these peripheral clusters of mitochondria, so that these
organelles are not near sites of Ca2+release from the SR,
but they are in close proximity to the capillaries. Indeed,
peripheral grouping of mitochondria in mammalian fibers
is mostly present in “red,” richly vascularized muscles.
These peripheral mitochondria may not “sense” the SR Ca2+
release, but are near oxygen sources. Unfortunately, although
the mitochondria content of more exotic mammal, such as
the cheetah known for the fastest running speed, has been
explored , no information of the actual positioning of
mitochondria relative to CRU is available for these muscles.
Two pieces of evidence indicate that the specific targeting
of mitochondria at the I band and their structural coupling
to CRUs in mammalian muscles are not due to chance.
First is the fact that association of mitochondria with
CRUs is acquired as a developmentally regulated event
during postnatal differentiation in mouse  and during
a recapitulation of these events in the recovery of human
. Secondly, strong connecting tethers link the junctional
SR of CRUs to adjacent mitochondria (Figures 2(a) and 2(c),
arrows) [22, 26]. The tethers link directly the SR membrane
to the outer mitochondrial membrane and seem to be strong
enough to hold them together if they are pulled apart (such
as if the fiber is exposed to hypotonic solutions).
An extensive mitochondrion-CRU association has been
detected in muscles from the domestic cat (unpublished
observations): from the guinea pig  and from human
muscles [21, 30, 31]. Taken together, these facts indicated
that a major subset of mitochondria in mammalian muscle
are in a specifically planned close structural and func-
tional proximity to the SR, and more specifically with
the domains (CRUs) that are responsible for Ca2+release
during excitation-contraction coupling. The association of
the selected mitochondria with CRUs is very extensive, since
approximately a quarter of the elongated mitochondrion
outer surface is in very close proximity to a jSR element and
is maintained by connecting tethers. This disposition seems
to be common to muscles from placental mammalian that
have been examined by ultrastructure.
It can also be inferred that the mitochondria that are
not tethered to CRUs may be free to move and perhaps
malemmal spaces. Their presence is driven to the proximity
of capillaries by the necessity of a high anaerobic profile.
Indeed, the highly aerobic diaphragm of the smallest mam-
mal, the shrew, is intensively vascularized and displays an
extensive array of peripheral mitochondria .
One notable exception to the specific mitochondrion-
CRU alignment in mammalian muscles is found in the
superfast cricothyroid muscle that produces the ultrasound
used by the bats for echolocation . In these muscles, the
mitochondria are not at the triads, but are located in long
longitudinal lines between the myofibrils, a disposition that
is typical of all nonmammalian vertebrates (see below).
Additionally, a muscle from a marsupial also shows no
specific relationship between mitochondria positioning and
CRUs (unpublished observations).
3.2. Birds. Birds, like mammals, are homoeothermic, and
some of them exhibit very fast and continuously active
muscles. It would be expected that if specific relationships
and capillaries were of overriding functional importance,
such relationships would be found in muscles from this
group. We were surprised to find that pectoral muscles in
domestic chicken and turkey (admittedly not very active
muscles), and also the leg and flight muscles of finches (that
are quite active) display mitochondria that are located in
single longitudinal columns between the myofibrils, with no
specific relationship to CRUs (Figure 3; see [34, 35]). Even
in the flight muscles of hummingbirds, mitochondria are
not especially associated with CRUs. It is likely however that
4 Journal of Biomedicine and Biotechnology
Figure 2: Higher magnifications highlighting association of mitochondria and SR (a)–(c). Tethering connections anchor mitochondria to
the jSR of the triads (CRUs) in images of mouse EDL ((a) and (c), white arrows). Similar tethers (white arrows) anchor mitochondria to the
longitudinal SR, not necessarily to CRUs, in the leg muscle from a finch (b).
the disposition of longitudinal mitochondria in the intermy-
ofibrillar spaces is not entirely random. Each mitochondrion
is in very close proximity to SR elements along its length,
and it seems to be anchored to them by tethers that greatly
resemble those in mammalian muscles (compare arrows
in Figure 2(b) with Figures 2(a)and 2(c)). We argue that
tethering to SR is responsible for holding the mitochondria
within the intermyofibrillar spaces and keeping them from
moving out and aggregating into subplasmalemmal clusters.
Longitudinal mitochondria have a chance of being at a
short distance from one or more CRUs along their length
as they run past the level of the sarcomere at which CRUs
are located (Figure 3(b), arrows). The frequency of such
encounters depends on the size of the mitochondrion and on
the frequency of triads along the T tubule network. Note also
that this configuration offers a very limited close proximity
between the mitochondrion surface and the CRU.
muscles of the finch is actually lower than in the mouse
leg muscle, despite the apparent similarity in the fast and
continuously active movements in these two species.
In the species mentioned above, there also seems to be
no accumulation of mitochondria in proximity of capillaries
even within the more aerobic muscles. Such accumulations
however are present in highly specialized muscles. The mito-
chondria respiration rates in the flight muscle of humming-
cles in mammals running at their maximum aerobic capaci-
ties. Capillary volume density is correspondingly higher, and
mitochondria are packed so highly that they almost compro-
mise the ability to produce force by crowding out myofibrils
clustered at a high density right at the capillary borders .
3.3. Reptiles. We examined the body muscles of two snakes
(Boa constrictor and Nerodia sipedon) and the leg muscles of
a lizard (Anolis carolinensis) as examples of reptile muscles
that are used for a variety of slow to fast movements.
The slower tortoise muscles have also been described .
Three different types of muscle fibres in Boa c. showed the
distinctive characteristics of tonic (large myofibrils, limited
membrane systems, and few mitochondria) probably used
when the snake crushes its prey, phasic “red” (smaller
myofibrils, slightly higher mitochondria content) probably
used when the snake moves around for longer periods of
time, and phasic “white” (smaller myofibrils, low mitochon-
dria content) probably used when the snake strikes a prey.
The lizard leg muscles had small myofibrils and frequent
graceful animals when in search of pray.
of mitochondria in keeping with the general motile pattern
of prolonged immobility alternated with brief periods of
fast activity. The overall distribution pattern is the same as
Journal of Biomedicine and Biotechnology5
Figure 3: Breast muscle from the finch shown in cross-sections (a)
and (b). In the finch, as in other birds, mitochondria are located
in single longitudinally oriented rows between the myofibrils. A
limited proximity between mitochondria and CRUs (arrows in (b))
occurs where the mitochondria cross the level at which T tubules
are located. Accumulations of mitochondria at the fiber edges are
not present in the flight muscles of many birds, indicating that the
mitochondria are not free to escape from the myofibrillar domains
of the fibers, probably due to tethering; see Figure 2.
in birds: and the same comments apply: mitochondria are
located in single columns occupying the intermyofibrillar
spaces, with no special relationship to CRUs and no accu-
mulation at capillaries (Figure 4, from the lizard).
As in the case of birds, a fast, continuously active muscle
provides an ultimate example of functionally designed orga-
nization. “Sound production is one of the most energetically
costly activities in animals” . Despite extremely reduced
tension production that minimizes contractile use of ATP
 and some other mechanical tradeoffs strategies , the
rattlesnakes tail shaker muscle is a fascinating example of the
adaptations necessary to produce and maintain extremely
fast contraction-relaxation rates . Mitochondria are
indeed at high density in these muscles and large clusters are
for the most part fairly close to capillaries, but they do not
bear a specific spatial relationship to CRUs .
3.4. Amphibia. In muscles of amphibia, as exemplified by
various species of Rana (R. pipiens, R temporaria [43, 44]; see
the large majority of the mitochondria tend to be located
between the myofibrils . Frog mitochondria are fairly
and an individual organelle may run for the length of one-
Figure 4: Longitudinal sections of leg muscles from a small lizard,
as an example from reptiles. The myofibrils are small, and the
CRUs (triads) are frequent, as expected from a rapid muscle.
Mitochondria are not frequent, and they are located in single
longitudinal rows between the myofibrils. An occasional proximity
to CRU occurs when the mitochondria cross the T tubule network
(arrows, see also Figures 3 and 5 for birds and amphibia).
two sarcomeres (Figure 5). There is no peripheral clustering
at the fiber edge. The disposition is the same in sartorius
and gastrocnemius where mitochondria are unusually scarce
(Figure 5) and in the iliofibularis where they are more
In general, none of the Rana muscles have a content of
mitochondria comparable to that of rat or mouse, indicating
a limited reliance on oxidative phosphorylation and there are
no descriptions of muscle with an extensive mitochondria
accumulation. Tonic fibers that sustain prolonged periods of
activity (e.g., those used in the mating amplexus) have an
even lower density of mitochondria, in keeping with the fact
that although the contractions are prolonged, they involve
very slow cycling cross-bridges and thus require a limited
amount of ATP.
3.5. Fish. At the lower end of the vertebrate subphylum,
we collected images from tail musculature of a variety of
fish, from primitive to more advanced. For example, see
[47–50]. All muscles fibers, including the “red” ones used
for powering swimming motion , contain elongated
mitochondria that are either located in longitudinal slits
between myofibrils (Figure 6(a)) or, more frequently, at the
fibers’ edges, under the plasmalemma (Figure 6(b)). An
interesting variation relative to birds, reptiles, and amphibia
is the fact that intermyofibrillar mitochondria are most
6 Journal of Biomedicine and Biotechnology
Figure 5: Images from the frog gastrocnemius (a) and sartorius (b)
in longitudinal sections. The predominant location of mitochon-
dria is similar to that of birds and reptiles, again providing limited
contacts with CRUs. Note overall scarcity of mitochondria in these
muscles gastrocnemius. Others have more frequent mitochondria
but still no large clusters at the periphery.
Figure 6: Cross-sections of small fibers in zebrafish. These images
illustrate two characteristics of mitochondria (m) disposition in
fish muscles. There is a tendency for clustering of the organelles in
small groups that are mostly segregated to the fibers’ edges. In this
manner, proximity of the mitochondrion’ surface to CRUs is quite
often, particularly if more numerous, collected into small,
longitudinally aligned clusters. This tendency is strongly
emphasized in the case of cold adaptation in fish such as
the striped bass, that results in an increase of mitochondrial
volume density by as much as 230% . While in the fish
exposed to warmer water the mitochondria are positioned
in single longitudinal rows between the myofibrils, in the
cold-adapted fish the mitochondria are accumulated in
large clusters both between the myofibrils and at the fiber
periphery. The direct effect of clustering is that of decreasing
the probability that an individual mitochondrion comes to
close proximity to a CRU. Higher oxidative capacity relates
to higher mitochondrial content, but not to a better contact
with CRUs [53, 54].
Interestingly, in superfast sound-producing swimbladder
musculature of Opsanus tau (toadfish), where the mitochon-
dria are not frequent [55, 56] and of Porichthys notatus (mid-
shipman) where they are quite abundant , mitochondria
are almost completely excluded from the regions of the fiber
containing myofibrils and placed instead in the central core
and outer subplasmalemmal ring. The physiology of these
fast-acting muscle fibers, like those of the rattlesnake, has
been modified for superfast repetitive contractions with little
This brief comparative look at skeletal muscles shows that
a well-regulated, close, extensive association between mito-
chondria and CRUs is present only in mammalian skeletal
muscles. In other vertebrate muscles, the mitochondria-
CRU proximity is far less extensive and less well regulated.
mammalian muscles is a relatively late event in evolutionary
times, it must confer some advantage, but it may not
be essential to muscle function. Apparently, coupling of
muscle activation to stimulation of mitochondrial aerobic
ATP production must occur even in the absence of the
It is hard to actually pinpoint the precise advantage
that extensive mitochondria-CRU association confers. Even
assuming that some Ca2+uptake by mitochondria is abso-
lutely essential, it must be remembered that this uptake
must be limited in order to avoid the total collapse of
the mitochondrion’s inner membrane potential. So, it is
not entirely clear that the extensive ER/SR-mitochondria
contacts that are present in liver and in mammalian skeletal
muscle are necessary and/or advantageous relative to the
more fleeting contacts between CRU and mitochondria of
muscles in lower vertebrates.
Perhaps the answer, in the case of mammalian skeletal
muscle, lies in the fact that mitochondria-CRU contacts
foster the differentiation of complex mitochondrial shapes.
Increases in cytosolic calcium levels above resting seem to
reduce mitochondrial motility , so an initial association
of large mitochondria with CRUs may have a positive feed-
back effect, enhancing and stabilizing further associations.
Mitochondria that are not tethered to SR and thus end up
in peripheral clusters are smaller and not as extensive as the
Journal of Biomedicine and Biotechnology7
CRU-associated ones. During postnatal muscle fiber matu-
ration in mouse, extension of mitochondria into branched
pattern and their association with CRUs occur in parallel
. A direct effect of this unique mitochondria tendency
towards extension for long distances both longitudinally
and transversely is the effective spread of information over
relatively large regions of the fiber, and this may have some
oxide flashes (mSOF) in resting and activated mouse FDB
fibers occur simultaneously over extended regions reflecting
the complex mitochondrial network . Considering that
a moderate level of superoxide production has physiological
effects (discussed in ), coordination of its production is
certainly of advantage.
It is, however, also noteworthy that extensive CRU-
mitochondria contacts are avoided in a mammalian fast-
acting muscle capable of prolonged activity: the bat cricothy-
roid, used for echolocation . Additionally, mitochondria
tethering is “loosened” in a mouse muscle with a leaky
mutation of the RyR  emphasizing the inherent risks of
a close proximity of mitochondria to Ca2+sources. Perhaps
fish muscles have the best solution. Mitochondria clustering
in those muscles has the effect of reducing the portion
of each organelle’s outline that faces directly towards a
CRU, thus reducing the influence of CRU’s Ca2+release on
mitochondria and allowing the development of some of the
fastest known muscle fibers, where mitochondria interfere
very little with Ca2+cycling.
T tubule: Transverse tubule.
Extensor digitorum longus
Flexor digitorum brevis muscle
The authors thank Dr. Angela Dulhunty from the Australian
National University for kindly providing a sample of Kanga-
roo muscle, Mr. Mathew Close for lizard and snake muscles,
and Mrs. Inna Martinyuk for electron microscopy support.
This work was supported by NIH Grant RO1 H 48093 to C.
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