ISSN 1990-519X, Cell and Tissue Biology, 2007, Vol. 1, No. 2, pp. 183–190. © Pleiades Publishing, Ltd., 2007.

Original Russian Text © S.A. Krolenko, S.Ya. Adamyan, T.N. Belyaeva, T.P. Mozhenok, A.V. Salova, 2007, published in Tsitologiya, Vol. 49, No. 2, 2007.

Confocal Microscopy Study of Membrane Organelles

of the Skeletal Muscle Fiber
in the Process of Zenker’s (Spreading) Necrosis
S. A. Krolenko, S. Ya. Adamyan, T. N. Belyaeva, T. P. Mozhenok, and A. V. Salova
Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia
e-mail: Krolenko@mail.cytspb.rssi.ru
Received May 17, 2006

Abstract—Using laser confocal microscopy and some vital fluorescent dyes (acridine orange, RH 414,
DiOC6(3), rhodamine 123, fluorescein dextran), changes of the T-system and cellular acidic organelles were
studied during spreading (Zenker’s) necrosis of isolated frog skeletal muscle fibers. The most characteristic of
the initial stages of development of Zenker’s necrosis is the formation of numerous vacuoles as a result of local
T-system swellings. The vacuole length can reach tens of micrometers. They are located both near nuclear poles
and between myofibrils. Until the moment of contraction knot separation, the vacuoles preserve their connec-
tions with normal T-tubules and under certain conditions (glycerol influx to the fiber) are reversible. The vacu-
oles deform nuclei and cisternae of the sarcoplasmic reticulum. Acidic cell organelles accumulating acridine
orange (lysosomes, late endosomes, trans-Golgi cisternae) are located in the immediate vicinity both of normal
and of vacuolated T-tubules. In the course of the development of the pathological process, the size and number
of acidic organelles increases and they tend to be clustered. Vacuolation of the T-system during necrosis was
not accompanied by vacuole content acidification. At late stages of necrosis, alterations of nuclei and sarcoplas-
mic reticulum were observed. The role of cellular acidic organelles and of the T-system vacuolation in devel-
opment of various muscle pathologies is discussed.

Key words: skeletal muscle fiber, spreading (Zenker’s) necrosis, T-system, vacuolation, acidic organelles, con-
focal microscopy, acridine orange, RH 414.
DOI: 10.1134/S1990519X07020101

Abbreviations: AO—acridine orange; SR—sarco-
plasmic reticulum; ZN—Zenker’s necrosis.
INTRODUCTION
The Zenker’s, or spreading, necrosis (ZN) of skele-
tal muscle fibers represents a fast (over several hours)
disintegration of the muscle cell in response to its local
injury. Morphological alterations occurring in the mus-
cle fiber during ZN are described in detail in the litera-
ture (Krolenko, 1975; Karpati and Carpenter, 1982;
Casademont et al., 1988; Carpenter and Karpati, 1989).
This necrosis is known to be accompanied by a local
myofibril contraction—a contraction knot is formed,
the shape of nuclei is changed, and numerous vacuoles
appear, with the origin of these vacuoles not always
being clear. Data on the state of acidic membrane
organelles (lysosomes, late endosomes, and trans-
Golgi cisternae) during ZN are quite limited (Duncan,
1987; Bechet et al., 2005).
Vacuolation is a very typical response of various
cells to damage. In skeletal muscle fibers, vacuolation
can be produced by osmotic shock, fatigue, and various
pathological processes (Krolenko, 1975; Krolenko et
al., 1995, 1998; Krolenko and Lucy, 2001, 2002). Ear-
lier, we studied in detail the vacuolation induced by the
efflux from muscle fibers of glycerol and some other
non-electrolytes after a preliminary loading of cells
with these substances (Krolenko, 1975; Krolenko et al.,
1995; Krolenko and Lucy, 2001). Vacuolation in these
experiments is due to a local swelling of transverse
tubules (the T-system). Under certain conditions, trans-
formation of the transverse tubules into numerous vac-
uoles is completely reversible and can be reproduced
many times in the same fiber.
Light optical and electron microscopy studies have
shown the T-system vacuolation to occur at certain
stages of ZN, however, other membrane organelles also
can be involved in the vacuolation processes (Krolenko,
1975). New possibilities of studying these processes
were provided by the use of confocal microscope in
combination with various vital fluorescent dyes. Pre-
liminary data on the distribution of acridine orange
(AO) in damaged fibers were obtained in our earlier
studies using a routine fluorescent microscope (Kro-
lenko et al., 2003).

183
184 KROLENKO et al.
The goal of the present work was to study localiza-
tion of acidic structures and their interactions with nor-
mal and vacuolized T-system, as well as to elucidate the
origin of the vacuoles appearing during muscle fiber
damage. To solve these problems, we used laser scan-
ning confocal microscopy of the frog living isolated
muscle fibers stained with fluorescent vital stains.
MATERIALS AND METHODS
The experiments were carried out on single fibers or
fiber bundles (2–3 fibers) isolated from m. iliofibularis
of the frog Rana temporaria in the winter and spring
seasons at 20–25°C. The composition of the Ringer
solution, the chamber construction, the procedure for
changing the solutions, and a number of other method-
ical details were described earlier (Krolenko et al.,
1995, 1998, 2003).
Incubation of fibers in Ringer solution with glycerol
(100 mM) for at least 30 min and subsequent washing-
out of the fibers from glycerol allowed us to obtain vac-
uolized fibers. The subsequent incubation of the vacu-
olized fiber in the Ringer solution with glycerol led to
the disappearance of vacuoles (Krolenko et al., 1995).
The presence of dyes in the solution had no effect on
the vacuolation and devacuolation processes.
In order to stain acidic membrane organelles, the
metachromatic dye AO (Merck) was used at a concen-
tration of 0.25 µg/ml. This dye emits in the green area
of the spectrum when in monomer form. It is accumu-
lated inside the acidic membrane vesicles according to
the proton gradient, and forms dimers and oligomers at
high concentrations, which leads to a fluorescence shift
to the red area of the spectrum (Palmgren, 1991; Schin-
dler et al., 1996; Clerc and Barenholz, 1998; Zelenin,
1999; Krolenko et al., 2006). The muscle fibers were
stained with AO for 40–100 min in a large Ringer solu-
tion volume, and then the fibers were transferred into an
observation chamber under the confocal microscope in
the presence of the dye. To stain the T-system, a lipo-
philic dye RH 414 (Molecular Probes) at a concentra-
tion of 15 µg/ml was used. This dye does not penetrate
the muscle fiber membrane and is used to stain sarco-
lemma and T-system tubules (Krolenko et al., 1995,
1998). The time of preliminary staining did not exceed
20 min.
In several experiments, fibers were simultaneously
stained with RH 414 and AO or with RH 414 and the
hydrophilic dye fluorescein dextran (10 000 MW,
Molecular Probes); at concentrations of 40–100 µg/ml
the fluorescein dextran did not penetrate the plasma
membrane (Krolenko et al., 1998). In addition, the flu-
orescent probes rhodamine 123 and DiOC6(3)
(Molecular Probes) were used at concentrations of
1−10 µg/ml. These probes penetrate the cell and stain
membranes of the sarcoplasmic reticulum (SR) and
mitochondria (Haugland, 2004). The staining took
10−30 min and then the dye was washed out prior to
microscopy. These dyes, used separately or in various
combinations, allowed observation of nuclei, sarco-
lemma, transverse tubules, myofibrils, and the majority
of membrane cell organelles in normal and damaged
muscle fibers.
A Leica TCS SL confocal fluorescent scanning micro-
scope (using an HCX PLAPO CS 63×/1.32 objective)
and BioRad 1024 (using a Plan Apo 60×/1.4 objective)
were used in the work. The observation was usually
performed in the superficial layers of the fiber
(10−15 µm). Serial sections (Z-series) were cut parallel
to the longitudinal fiber axis. In the Z-series,
10−20 images were consecutively recorded using steps
of 0.3–0.5 µm. For each image, 4–6 runs of the scan-
ning ray were used.
Stained fiber fluorescence was excited using argon
or krypton/argon lasers (in the spectral regions of
458 nm and/or 633 nm). The laser power did not exceed
30% of its maximal value. The fluorescent emission
during the fiber staining with AO, AO + RH 414, and
AO + fluorescein dextran was recorded simultaneously
in the green (500–560 nm) and red (590–680 nm) chan-
nels. For other dyes, optic filters corresponding to the
area of the maximal dye emission were used. The
images obtained with the confocal microscope were
processed using the Leica TCS SL, Image J, and Photo-
shop programs.
For electron microscopy, fixation with 1% glutaral-
dehyde in the usual Ringer solution was used, with sub-
sequent postfixation with 1% OsO4. The specimens
were embedded in araldite.
RESULTS AND DISCUSSION
Normal muscle fibers. Simultaneous staining of
living muscle fibers with AO and RH 414 was rather
informative: Rh 414 allowed the identification of
plasma membrane, normal and vacuolized transverse
tubules, while AO stained cell nuclei and accumulated
as granules in acidic membrane organelles (lysosomes,
late endosomes, and Golgi cisternae) (Fig. 1a). The
main portion of the sarcoplasm displayed a weak green
fluorescence, on the background of which the more
intensively stained A-bands could be identified
(Fig. 1b). Binding of AO with muscle myosin was
noted earlier (Kalamkarova et al., 1965; Gamaley and
Kaulin, 1988). The AO granules were always present at
the nuclear poles and were also located throughout the
entire fiber among myofibrils as short parallel rows
often composed of 2–3 granules located in neighbor
sarcomers (Krolenko et al., 2006).
Measurement of the granule sizes in serial optical
sections showed them to have a shape close to spheri-
cal. The granule diameter was 0.4–1.5 µm. The number
of granules per 15 fibers, was, on average, 80 ± 5 per
10000 µm2 of the optical section surface. This value
varied, not only in different fibers, but also in different
areas of the same fiber. For example, in serial sections
CELL AND TISSUE BIOLOGY Vol. 1 No. 2 2007
 CONFOCAL MICROSCOPY STUDY OF MEMBRANE ORGANELLES 185

(‡) (e)

(b)
(f)

(c)

(g)

(d) (h)
Fig. 1. Confocal images of the normal and damaged muscle fiber. (a) Location of T-tubules and acidic membrane structures in the intact
muscle fiber. The fiber was stained simultaneously with AO and RH 414. Merge image of green and red channels. The T-system is seen as
regularly arranged dotted lines perpendicular to the long fiber axis and located at a distance of a sarcomere length from one another (in our
experiments, about 2.2 µm). AO granules are located near nuclear poles and between myofibrils, where they form pairs, triplets or short
longitudinal rows. Most AO granules are adjacent to transverse tubules. (b) Damaged muscle fiber at a distance from ZN boundary. AO
staining (merge green and red images). Light green transverse lines correspond to myofibril A discs. T-system vacuoles are revealed as
longitudinal unstained structures, often with AO granules located at their boundaries. (c) A strongly vacuolized part of the muscle fiber
between two contraction knots located out of the vision field of the present image. Simultaneous staining with AO and RH 414 (merge of
green and red images). Fusion and tight contacts of neighbor vacuoles and their vicinity with AO granules. Fine preservation of normal
transverse tubules. (d) Location of AO granules in damaged muscle fibers. AO staining (merge green and red images). The upper fiber—
near the ZN boundary. The lower fiber—contraction knot containing numerous deformed nuclei and AO granules. (e) Location of AO gran-
ules and the T-system in damaged muscle fibers. Simultaneous staining with AO and RH 414 (merge of green and red images) with sub-
sequent incubation of the fiber in monencine (50 ng/ml) that resulted in 20 min in the disappearance of most AO granules. Arrows indicate
necrosis boundary in the upper fiber. Small vacuole is adjacent to the lateral surface of swollen nucleus. A vessel and completely necrotized
brightly stained fiber visible in lower part of the image. (f–h) The ZN boundary. Simultaneous staining with fluorescein dextran (MW 10000)
and RH 414. (f) Confocal image in the green channels (fluorescein dextran). (g) Confocal image in the red channel (RH 414). (h) Merged
image of g and h. All vacuoles contain extracellular marker—fluorescein dextran. The vacuoles acquire a spherical shape and sometimes
preserve a paired arrangement in the area of contraction knot separation (arrow). Scales—10 µm, arrows—necrosis boundary.

CELL AND TISSUE BIOLOGY Vol. 1 No. 2 2007

186 KROLENKO et al.
(‡)
(b)
(c)
(d)
(e)
Fig. 2. The T-system state of the damaged muscle fiber. The
fiber was damaged in the presence of RH 414. (a) Confocal
image directly on the ZN boundary. (b–e) At a distance from
the necrosis boundary of 0.2, 0.4, 1.0, and 2.0 mm, respec-
tively. Scale—10 µm, arrows—necrosis boundary.
of the fiber area, the number of granules in individual
sections varied by 1.5–2 times. We observed that the
acidic cell organelles in skeletal muscle fibers were in
close contact with transverse tubules (Fig. 1a). The
granules were adjacent to the T-tubule membranes but
did not fuse with them. Contacts between acidic
organelles and the transverse tubule membranes were
also preserved in the process of the T-system vacuola-
tion caused by glycerol removal from the fibers.
AO accumulated in the lumen of acidic membrane
vesicles according to the proton gradient, which was
indicated by the disappearance of AO granules under
(‡)
(b)
Fig. 3. Reversible vacuolation of the T-system during ZN.
The fiber was stained with RH 414. (a) Vacuolated part of
the damaged muscle fiber (3 h after beginning of necrosis).
Location of vacuoles marks nuclear location (arrow).
(b) The same area of the fiber after 10 min of incubation in
Ringer solution with glycerol (100 mM). Most vacuoles
decreased in size or disappeared.
the effect of the ionophore monencine (Fig. 1e) (Kro-
lenko et al., 2003, 2006). The degree of AO accumula-
tion also depended on many other factors, for example,
the permeability of vesicular membranes, aggregation
of AO molecules, osmotic properties of vesicles, etc.
(Weisz, 2003). When concentrated in the lumen of
these organelles at low concentrations in monomer
form, AO emitted in the green area of the spectrum,
while at higher concentrations it formed dimers and oli-
gomers with an emission maximum located in the red
area of the spectrum. Acidic cell organelles emitted in
both the green and in the red areas of the spectrum in
most cases, because they contained AO monomers,
dimers, and oligomers in various ratios. Taking the fact
into account that the content of lysosomes and late
endosomes is the most acidic material in the cell,
(Weisz, 2003) these organelles undoubtedly partici-
pated in the formation of AO granules. Literature data
on the disperse distribution of Golgi apparatus in skel-
etal muscle fibers (Ralston, 1993; Lu et al., 2001; Ral-
ston et al., 2001) also suggest that that the Golgi appa-
ratus made a significant contribution to the formation of
AO granules.
Zenker’s necrosis. Under our conditions, the devel-
opment of ZN, and, in particular, the number and the
formation rate of contraction knots and degree of their
separation from the rest of the fibers (the ZN bound-
ary), varied significantly. The observations were usu-
ally performed in the area of separation of the contrac-
tion knot and far from it (Figs. 1, 2). In several experi-
ments, the changes in the same fiber were studied at
various distances from the ZN boundary, up to the area
CELL AND TISSUE BIOLOGY Vol. 1 No. 2 2007
 CONFOCAL MICROSCOPY STUDY OF MEMBRANE ORGANELLES 187

which was nearly normal (Fig. 2). The most typical

morphological change in the fiber located far from the
ZN boundary, which preceded formation of the con-
traction knot, was vacuolation (Figs. 1b–1h, 2). Use of
the T-system membrane marker RH 414 clearly demon-
strated the origin of these vacuoles from the transverse
tubules. (‡)
The pattern of vacuolation during ZN, especially at
some distance from the necrotic area, on the whole was
rather similar, with vacuolation occurring during the
late stages of washing-out glycerol or upon its loading
and removal in repeated cycles (Krolenko et al., 1995,
1998; Krolenko and Adamyan, 2000). Usually, large
vacuoles (more than 2 µm) which were located at cell
nuclear poles and which formed long longitudinal
chains between myofibrils were predominant. Vacuoles
in the chains preserved their individuality, tightly
opposed to each other. More seldom, fusion of neighbor
vacuoles into long (tens of micrometers) flat cisternae
(Figs. 1b, 1c, 2) occurred. The presence of such huge
vacuoles was also characteristic of the spontaneous
vacuolation which was occasionally observed on fibers
without obvious signs of mechanical injury and ZN. (b)
Unlike the vacuole formation during primary washing-
out of glycerol, in ZN no appearance of numerous small
vacuoles with gradual increase of their size was
observed (Krolenko et al., 1995; Krolenko and
Adamyan, 2000).
Vacuolation of muscle fibers far from the ZN bound-
ary was not accompanied by the disappearance of nor-
mal transverse tubules (Fig. 2d, 2e). The T-system
could be well preserved, even in short fragments of
almost completely disintegrated fibers (Fig. 1c) and
sometimes directly at the ZN boundary (Fig. 1e). How-
(c)
ever, in most cases, arrangement of vacuoles at the ZN
boundary became chaotic and normal transverse
tubules are not seen (Figs. 1d, 1h, 2a, 2b). The fiber vac- Fig. 4. Location of T-system vacuoles near the nuclear
poles. (a) Confocal image of the damaged muscle fiber.
uolation accompanying development of ZN was, as a Simultaneous staining with AO and RH 414 (merge green
rule, completely or partly reversible under the effect of and red images). Large vacuoles are located near the nuclear
glycerol influx (Fig. 3). In our opinion, this indicates poles and deform the nucleus. The AO granules are located
preservation of contacts between vacuoles and the nor- between vacuoles. Scale—5 µm. (b, c) Electron microscope
mal areas of the T-system (Krolenko, 1975; Krolenko structure of the damaged fiber far (b) and near (c) Necrosis
boundary. Vacuoles are pressed into the nucleus. Scales—
and Lucy, 2001). It is to be noted that the morphologi- 1 µm.
cally straight connections of large vacuoles with nor-
mal tubules were practically unresolved; however, the
use of extracellular markers able to fill transverse perimeter. In the same perinuclear areas, vacuoles were
tubules (ferritin, high molecular weight fluorescein the last to disappear in the process of devacuolation.
dextran) clearly demonstrated the existence of such The fiber areas near the nuclear pole seem to have a less
contacts (Krolenko, 1975; Krolenko et al., 1995; Kro- dense packing than the remaining fiber portion filled
lenko and Adamyan, 2000). Probably, a certain degree with regularly arranged myofibrils. This provides opti-
of integration of vacuoles with residues of normal mal conditions for the appearance of vacuoles in this
tubules can be preserved up to the ZN boundary, as vac- particular area. Formation of vacuoles near nuclei
uoles in this area were revealed even in the case that the seemed to be facilitated by organization of transverse
RH 414 staining was performed after ZN development tubules in the form of glomerules (Voigt and Dauber,
(not shown). 2004). The use of RH 414 showed beyond doubt that
During ZN, and particularly during spontaneous these perinuclear vacuoles originate from the T-system.
fiber vacuolation, vacuoles appear primarily at nuclear Usually the perinuclear vacuoles were less compressed
poles; more seldom they surround nuclei at the entire than the vacuoles located between myofibrils. Nuclear

CELL AND TISSUE BIOLOGY Vol. 1 No. 2 2007

188 KROLENKO et al.

(‡)

(b)

Fig. 5. Changes of the DiOC6(3) staining in the process of

ZN. (a) The ZN boundary. (b) Distant from the necrosis
boundary. Scale—10 µm.

deformation by vacuoles was typical, which was clearly

observed to result in nuclear poles becoming enlarged
and concave, both in living cells and in fixed prepara-
tions (Figs. 1e, 4). Such nucleovacuolar interrelations
indicate that vacuoles represent a more “rigid” mem-
brane structure than nuclei. The mechanical properties
of vacuoles could be due to both purely osmotic factors
and the physical properties of their membranes.
During ZN, distribution of AO in vacuolized fiber
areas continued without essential changes, and on the
whole was similar to that in experiments with reversible (c)
T-system vacuolization caused by glycerol washing-out
(Krolenko et al., 2006). A small increase in sarcoplasm
background staining was noticed in the green area of Fig. 6. Changes of the rhodamine 123 staining in the pro-
cess of ZN. (a) The ZN boundary; (b) Near the necrosis
the spectrum. Aside from the more intensively stained boundary. (c) Distant from the necrosis boundary. Scales
myofibril myosin, longitudinal bands appeared 10—µm.
between myofibrils and near nuclear poles (Fig. 1b, 1d,
1e). In these bands, structures of irregular or oval shape
could sometimes be identified. Their intensity of stain- decrease of the AO concentration in the granules. Use
ing was significantly lower than that of the AO gran- of the laser scanning confocal microscope showed that
ules. There was a trend toward an increase in the num- under normal conditions, both the red and the green
ber and size of the AO granules, disturbance of their components were present in practically all granules
ordered arrangement in the sarcoplasm, and their clus- (Krolenko et al., 2006). In the present study, no
tering (the location of several granules in close contact marked changes of the character of the granule AO
with each other). fluorescence were revealed during ZN. As in the case
In our previous work (Krolenko et al., 2003) on the of vacuolation caused by glycerol efflux (Krolenko
AO distribution in the muscle fiber, which was carried et al., 2006), the AO granules during ZN were in a
out with use of a routine fluorescent microscope, we close contact with normal and vacuolized T-tubules.
found an increase of the green component of the AO Adjustment of several AO granules to the walls of one
granule fluorescence in damaged muscle fibers, i.e., a vacuole or the location of granules between neighbor-

CELL AND TISSUE BIOLOGY Vol. 1 No. 2 2007

 CONFOCAL MICROSCOPY STUDY OF MEMBRANE ORGANELLES
ing vacuoles (Figs. 1c, 1d, 4a) was typical. In the pro-
cess of ZN development, neither fusion of AO granules
with vacuoles, nor AO accumulation in the vacuole
lumen were observed, which may have indicated acidi-
fication of their content. The conversion of vacuoles
into lysosomes or similar structures, which has been
suggested in some studies, (Libelius et al., 1979a,
1979b; Bird et al., 1981; Vult von Steyern et al., 1996)
seems to be possible only during slowly developing
pathological muscle fiber changes.
Of particular interest during ZN was the behavior of
nuclei, vacuoles, and acidic cell organelles in the zone
of active muscle fiber disintegration, specifically in the
regions of the contraction knot and its separation from
the remaining, not yet necrotized, part of the fiber
(Figs. 1d–1h, 2a). In these areas, single AO granules,
vacuoles, and nuclei were still preserved, but the shape
of all these structures became essentially changed.
Nuclei lost their cigar-like shape, swelled, and twisted,
while vacuoles acquired a spherical shape. Among such
predominantly chaotically distributed vacuoles, vacu-
oles arranged in pairs were present (Figs. 1f–1h), which
indicates some physical or biochemical mechanisms of
the coupling of neighbor vacuoles in the muscle fiber.
There were no morphological signs of preservation of
normal transverse tubules in this area. According to the
distribution of fluorescein dextran fluorescence, vacu-
oles in the area of formation and separation of the con-
traction knots represent completely closed membrane
vesicles.
The staining of the fiber with lipophilic permeable
dyes (DiOC6(3), or rhodamine 123) demonstrated
essential changes of the SR and mitochondria during
ZN (Figs. 5, 6). In intact fibers, DiOC6(3) revealed dis-
tinct transverse striations (Fig. 5b) that corresponded to
distribution of SR elements along the sarcomeres (Kro-
lenko et al., 1995). A similar picture was observed in
staining with rhodamine 123 (Fig. 6c). As well, both
dyes stained longitudinal bands in the interfibrillar
space, which seemed to be due to accumulation of the
dyes in mitochondria arranged in chains. The fiber
areas adjacent to nuclear poles were also stained. These
dyes did not stain the nuclei, sarcolemma, and either the
normal or the vacuolized T-system. The development of
ZN was accompanied primarily by changes in the pat-
terns of transverse striation: this became more hetero-
geneous and was gradually transformed into numerous
bright bodies, 1–2 µm in diameter (Figs. 5a; 6a, 6b).
They were usually longitudinally oriented, but could
also be arranged perpendicular to the long axis of the
fiber. We consider this phenomenon to be the result of
the swelling and fusion of SR elements, primarily the
terminal cisternae (Krolenko, 1975). The contribution
of swelling to the topography of such structures could
also be made by deformation of the SR elements by
vacuoles formed from the T-system and appearing as
unstained cavities (Fig. 6b). Directly on the ZN bound-
ary, the total sarcoplasm staining and the number of
brightly stained bodies increased (Figs. 5a, 6a, 6b). The
CELL AND TISSUE BIOLOGY Vol. 1 No. 2 2007
189
necrotized fiber areas appeared to be structureless,
which indicated a fast lysis of cell membrane organelles
stained with lipophilic dyes.
Our results indicate that T-system vacuolation is one
of the primary and most obvious morphological
changes of muscle fibers during ZN. The cellular and
molecular mechanisms of the T-system vacuolation
have not yet been elucidated; however, vacuole forma-
tion most probably results from the efflux of ions and
metabolites from the damaged cell (Krolenko, 1975;
Kao and Gordon, 1977; Corbett, Pollock, 1981; Casa-
demont et al., 1988; Krolenko and Lucy, 2001). Such a
mechanism was confirmed by the similarity of the data
on vacuolation processes during the muscle fiber dam-
age with the results of experiments in which glycerol
was washed out after its initial loading. Under certain
conditions, ZN development can terminate and be
replaced by regenerative processes (Bechet et al.,
2005). In this case, as well as during several slowly
developing pathological processes, vacuoles can persist
in fibers for a long period. Formation of vacuoles,
including the autophagic ones, is characteristic of many
forms of hereditary and acquired myopathies (Engel,
1970; Kao and Gordon, 1977; Nonaka and Sugita,
1981; De Bleecker et al., 1993; Bechet et al., 2005).
The etiology of these myopathies has often been
unclear. We believe that the ability of the T-system to
easily form large vacuoles under the effect of the efflux
of various substances could be directly related to some
types of pathological vacuolation.
ACKNOWLEDGMENTS
The work was supported by the Russian Foundation
for Basic Research (project 04-04-49394) and in part
by the Joint Research Center “Materials science and
characterization in high technologies”.
We thank Drs. M.D. Fadeeva, Yu.M. Rosanov and
G.I. Shtein for their helpful advice and discussion.
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