www.elsevier.com/locate/micron

Micron 38 (2007) 659–667

Formation of leptofibrils is associated with remodelling of

muscle cells and myofibrillogenesis in the border

zone of myocardial infarction

Eduard I. Dedkov a, Alexander A. Stadnikov b, Mark W. Russell c, Andrei B. Borisov c,*a Department of Anatomy and Cell Biology, University of Iowa, Iowa City, USA

b Orenburg State Medical Academy, Orenburg, Russiac Division of Pediatric Cardiology, Department of Pediatrics, University of Michigan Medical School, Ann Arbor, USA

Received 19 April 2006; accepted 31 August 2006

Abstract

Leptofibrils, or leptomeres, remain the least studied cytoskeletal structures in muscle cells, and their function and mechanism of assembly are

still poorly understood. Our ultrastructural study of the surviving cardiac myocytes located in the perinecrotic border zone of the infarcted left

ventricle in rats revealed intense formation of leptofibrils and leptofibrillar clusters during 4–15 days following experimental myocardial infarction.

In the perinecrotic myocytes, leptofibrils developed predominantly in the subsarcolemmal areas, near disassembled intercalated discs and at the

sites of intense myofibrillogenesis in the peripheral zones of the sarcoplasm. We found that the development of these structures occurred before or

at the time of assembly of myofibrils. In our material, leptofibrils consisted of longitudinally oriented filamentous bundles inserted in electron dense

Z-band-like material and periodically crossed by 3–8 bands of this material with the period of cross-striation of 120–210 nm. The presence of

leptofibrils in growing cytoplasmic processes and ruffles developing in the border zone in the areas of lost intercellular contacts indicates their

formation de novo during post-infarction period. We observed four major morphological types of localization of these structures: (1) direct contact

of one end of leptofibrils with Z bands of nascent, mature or disassembling myofibrils; (2) direct contact with the sarcolemma: (a) multifocal

attachment of leptofibrils to the sarcolemma through the lateral surfaces of their minute Z band-like structures; (b) attachment of one or both ends

of leptofibrils to the sarcolemma without contacts or in contact with myofibrils; (3) attachment of leptofibrils to subsarcolemmal accumulations of

electron dense Z-band material in newly formed fasciae adherentes of the remodeled intercalated disks; (4) clustering and contacts of leptofibrils

with one another predominantly at the level of their Z bands. Interestingly, most leptofibrils of all four types were topographically associated with

the system of T-tubules, the sarcoplasmic reticulum and subsarcolemmal vesicles. Serial sections through the areas containing leptofibrils indicate

their spindle-like or nearly cylindrical shape. Thus, we found that leptofibrils assemble in terminally differentiated cardiac myocytes following

destabilization of their differentiated state and partial dedifferentiation induced by myocardial infarction. The results of this study demonstrate that

formation of leptofibrils, earlier described mainly in the developing and malignant muscle, is temporally associated with adaptive structural

remodelling and the activation of myofibrillogenesis in functionally overloaded cardiac myocytes of adult animals. Our findings suggest that re-

expression of some structural characteristics of the embryonic muscle appear to represent one of the mechanisms that underlie adaptive plasticity of

the myocardium following injury and under conditions of hyperfunction.

# 2006 Elsevier Ltd. All rights reserved.

Keywords: Myocardium; Injury; Cardiac myocytes; Leptomeres; Myofibrillogenesis; Leptofibrils; Myocardial infarction; Heart

1. Introduction

Leptofibrils (from Greek leptos meaning small, thin, fine and

Latin fibra, fiber), are still one of the least studied structural

* Corresponding author at: Room 8303A, MSRB III, Division of Pediatric

Cardiology, Department of Pediatrics and Communicable Diseases, University

of Michigan Medical School, Ann Arbor, MI 48109, USA. Tel.: +734 647 9429;

fax: +734 615 1386.

E-mail address: aborisov@med.umich.edu (A.B. Borisov).

0968-4328/$ – see front matter # 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.micron.2006.08.006

elements associated with the contractile system of cardiac and

skeletal muscle cells. Due to the very small size of these

structures, electron microscopy remains the only technique that

permits their reliable detection and characterization (for

literature, see Martynova and Borisov, 1987; Ghadially,

1997). Electron microscopy identifies leptofibrils as tiny

cross-striated myofibril-like structures that consist of long-

itudinally aligned bundles of 4–10 nm filaments periodically

crossed by 3–20 electron-dense bands. These dense bands

E.I. Dedkov et al. / Micron 38 (2007) 659–667660

resemble minute Z-bands of sarcomeres and are typically

located with a periodicity of 110–250 nm in different animal

species. Leptofibrils were first described in the growing skeletal

muscle latissimus dorsi anterior of young thrushes (Ruska and

Edwards, 1957). Further studies revealed the presence of

similar structures in embryonic skeletal and cardiac muscle of

birds and mammals (Bogusch, 1975; Adal, 1977; Myklebust

and Jensen, 1978; Hulland, 1988; Hosokawa et al., 1994) and in

the tumors of myogenic origin such as rhabdomyomas and

rhabdomyosarcomas (Fenoglio et al., 1976, 1977; Bleisch and

Kraus, 1980; Carstens and Martin, 1986; Kim et al., 1989;

Tanimoto and Ohtsuki, 1996). The presence of leptomeres has

been recently described in the sonic muscle of the marine

teleost fish Porichthys notatus (Nahirney et al., 2006). The

functional significance and the mechanisms of assembly of

leptofibrils still remain unknown. Up to the present time, there

is no well-established term for naming these structures.

Different authors have referred them to as supernumerary

striations of Z-line material, leptomeric myofibrils, leptomeric

fibrils, microladder, l-fibrils, fibrous banded structures, short

periodical bodies, zebra bodies, leptomeric organelles, pro-

dromal pattern of contractile material and leptofibrils (for

discussion, see Martynova and Borisov, 1987; Ghadially, 1997).

In this paper, we use the last term as it is the most accurate and

laconic.

In our earlier studies, we found a significant presence of

leptofibrils in cardiac myocytes isolated in culture from the

hearts of 2- and 14-day-old rats (Martynova and Borisov, 1987,

1990). The abundance and variety of structural modifications of

leptofibrils found in rat cultured cardiac myocytes exceeded

considerably the frequencies of their occurrence that we have

observed during nearly twenty years of electron microscopic

investigation of mammalian cardiac and skeletal muscle tissue.

Numerous leptofibrils and leptofibrillar complexes scattered

about peripheral areas of the cytoplasm were later described in

cultured feline cardiomyocytes (e.g. Figs. 4 and 6 in Simpson

et al., 1993) and in rat cardiac myocytes after exposure in

culture to the tumor promoter, phorbol ester (Nag and Lee,

1990). Sussman et al. (1998) recently revealed the presence of

leptofibrils and disarray of sarcomeric actin filaments in

cultured neonatal cardiac myocytes that were transfected with

recombinant adenoviral vectors to induce overexpression of

tropomodulin.

Leptofibrils are rarely found in terminally differentiated

cross-striated muscle tissue in vivo (for consensual opinion, see

comments on the report of Arbustini (1988) and Sage and

Jennings (1989)). This may indicate that the establishment of

the terminally differentiated state is associated with the

downregulation of leptofibril formation and the disassembly

or resorption of some of these structures. For these reasons, we

concluded that the assembly of leptofibrils represents a

recapitulation of a structural characteristic of the embryonic

phenotype during adaptive remodeling and redifferentiation of

cardiac myocytes in vitro (Martynova and Borisov, 1987).

Taking into account a significant plasticity of the differentiative

properties of cardiomyocytes in vitro (for literature and

duscussion, see Bugaisky, 1991; Borisov, 1991, 1998; Nag

et al., 1996), such an interpretation is plausible. However,

causal analysis of the interrelations between instability of the

differentiated state and active adaptive remodeling in cell

culture is very difficult. We should take into account some

specific conditions of the in vitro model that include interaction

of cells with an artificial substrate, loss of intercellular contacts

and normal cell–matrix interactions, as well as the absence of

vascularization and neuro-humoral control. Therefore, it has

remained unclear whether the formation of leptofibrils in

cultured cardiac myocytes was an artifact of the in vitro model,

or this process was really associated with adaptive reorganiza-

tion of cardiac myocytes and adaptive modulations of their

differentiative characteristics. To answer these questions, we

searched for an experimental model suitable for studies of

structural remodeling and reactivation of myofibrillogenesis in

cardiac muscle cells in vivo. The pathogenesis and the

dynamics of healing of left ventricular myocardial infarction

and compensatory plasticity of cardiac myocytes in rat

experimental model are well-characterized in the literature

(reviewed by Rumyantsev, 1991). There is convincing evidence

of a profound reorganization and compensatory hypertrophic

response of surviving muscle cells in the perinecrotic area

following mycardial infarction (for literature, see Rumyantsev,

1991; Cox et al., 1991). For these reasons, we have chosen this

experimental model to investigate whether destabilization of

the differentiative characterisctics of cardiac myocytes in vivo

is correlated with formation of leptofibrils. The studies of

structural characteristics of leptofibrils in different models of

cardiac and skeletal muscle adaptation and pathology will

provide new insights into understanding their functional role

and the mechanism of formation.

The purpose of the present study was to investigate whether

formation of leptofibrils occurs in the infarcted heart. To answer

this question, we studied the areas of compensatory remodeling

and hypertrophic growth of cardiomyocytes in the border zone

that surrounds the site of myocardial injury. To this end, special

attention was focused on the following three stages of the post-

infarction response of cardiac muscle cells: (1) destabilization

of their differentiated phenotype; (2) structural reorganization;

and (3) redifferentiation.

2. Materials and methods

2.1. Animal model

The experimental myocardial infarction of the left ventricle

was induced in adult 6- to 8-month-old rats by ligation of the

left coronary artery under ether anesthesia according to the

technique of Selye et al. (1960). The hearts of intact animals of

the same age were used as controls. Animals were euthanized

under ether anesthesia 3, 7, 15 and 21 days after infarction and

the hearts were excised. The portions of the left ventricular wall

of operated animals containing the area of infarction and

portions of normal control left ventricular tissue were rapidly

fixed by immersion in ice-cold solution of 4% parapformalde-

hyde and 2.5% glutaraldehyde in 0.1 M isoosmotic phosphate

buffer, pH 7.4 for 4 h. The tissue samples taken from the border

E.I. Dedkov et al. / Micron 38 (2007) 659–667 661

zone were cut into several pieces and fixed for an additional 4 h

at 4 8C in fresh aliquots of fixative.

2.2. Electron microscopy

After removal of the fixative, the samples were washed in 3

changes of 0.1 M phosphate buffer; each wash was for a period

of 15 min. The tissue was postfixed with 1% OsO4 in 0.1 M

phosphate buffer for 1.5 h at 4 8C, washed again in the buffer

solution 3 times (15 min each wash) and dehydrated through

graded ethanol series to absolute ethanol and acetone. After

infiltration with Epon/Araldite the samples were embedded in

Epon/ Araldite medium (Eponate 12-Araldite 502 kit, Ted Pella

Inc). After polymerization of the medium in blocks, sections

were prepared using a Reichert–Young Ultracut E ultramicro-

tome and mounted on grids. Following contrast staining with

uranyl acetate and lead citrate, the sections were examined with

a Philips CM 100 electron microscope at an accelerating

voltage 60 kV. Serial ultrathin sections were placed on the same

grid and identified for mutual matching at low magnification

2300�. Then the same areas were localized in neighboring

sections at higher magnifications which allowed us to detect the

same structures at different levels of sectioning.

3. Results

Following ligation of the left coronary artery, myocardial

infarction developed in the free wall of the left ventricle. The

necrotic injury typically included large areas of the sub-

endocardial zone and sometimes spread in the direction of the

subepicardial zone. Granulation tissue started to form and

replace the necrotic muscle tissue 2–3 days after infarction. By

that time we found the first clear manifestations of structural

remodelling in surviving cardiac myocytes located in the

perinecrotic zone. Muscle cells at the edge of the border zone

started to develop finger-like cytoplasmic processes located on

their free ends. At this stage, the areas of former intercalated

discs lost their characteristic ‘‘stair-step’’ contours. During the

next 3–10 days, the process of remodeling of the cell periphery

resulted in development of lamellopodia-like ruffles that were

gradually filled by nascent myofibrils and non-striated

cytoskeletal and filamentous structures (Figs. 1 and 2Fig.

1A-D and 2A,B). These newly formed cytoplasmic extensions

frequently contained leptofibrils and leptofibrillar complexes

that were either directly attached to nascent or partially

resorbed myofibrils or were located at close distance from the

sarcolemma (Figs. 1A–D and 2A–B). The length of leptofibrils

in our material varied between 0.4 and 1.3 mm and the width

was 0.1–0.45 mm. The period of their cross-striation typically

was 120–210 nm. Sometimes leptofibrils directly contacted the

inner surface of the sarcolemma without any structural

association with myofibrils (Fig. 2A–C). The resorption of

the contractile system and the intensity of remodeling of the

peripheral areas of the sarcoplasm greatly varied in individual

cells. For this reason, by the second week following infarction

myocytes in the perinecrotic zone represented a structurally

heterogeneous population of cells that underwent more or less

advanced processes of dedifferentiation and redifferentiation.

We observed leptofibrils mostly in cardiomyocytes containing

myofibrils with a developed sarcomeric organization which

exhibited manifestations of activated myofibrillogenesis.

When growing cytoplasmic processes of neighboring cells

met one another, they usually established contacts either along

their lateral surfaces (Fig. 3A–D) or end-to-end through their

distal tips (Fig. 4A,B). We observed the development of

leptofibrils in the subsarcolemmal areas of two adjacent cells

exactly at the sites of their contacts (Fig. 3A–D). Interestingly,

the electron-dense Z-band-like structures of leptofibrils of the

contacting myocytes were spatially adjusted and located

directly opposite one another (Fig. 3A–C). Another interesting

detail shown in Fig. 3B and C is attachment of Z-band material

of myofibrils to leptofibrils located on the inner surface of the

sarcolemma. This shows that leptofibrils may serve as transient

structures that serve as temporary anchorage for the attachment

of myofibrils to the sarcolemma. We did not find leptofibrils in

the atrial myocardium, and they were very rare in the right

ventricle and in the areas of left ventricle located outside the

perinecrotic zone.

Progressive development and maturation of newly formed

intercellular contacts result in re-establishment de novo of

fasciae adherentes between adjacent remodelled myocytes

(Fig. 4A and B). These areas represent one more typical site of

localization of leptofibrils. Leptofibrils located near the nascent

intercalated discs are either found near nascent myofibrils

without any contacts with the fasciae adherentes (Fig. 4A) or

directly connected to subsarcolemmal accumulations of Z-band

material on the inner surface of the sarcolemma (Fig. 4B). As

one can see in Figs. 1–4, leptofibrils are typically associated

with accumulations of subsarcolemmal vesicles, caveolae and

tubular elements of the sarcoplasmic reticulum. These vesicles

are surrounded by a single membrane, and the analysis of their

structure at a high magnification did not reveal any electron-

dense secretory product within these structures (Fig. 4A,B).

Their frequent close topographical association with the inner

surface of the cell membrane and the presence of multiple

caveolae-like openings on the sarcolemma (e.g. Figs. 1C–D,2B,

3B, 4A,B) may indicate the involvement of these structures in

the process of membrane biogenesis and consider them as the

source for assembly of the sarcolemma, the sarcoplasmic

reticulum and T-system. Thus, these structures appear to

represent a morphological manifestation of membrane growth.

This growth may result from the insertion of new membrane

material into the sarcolemma, which is necessary for the

formation of cytoplasmic projections, lamellopodia and ruffles.

Observations of the same leptofibrils sectioned longitudin-

ally in 2–3 serial ultrathin sections demonstrate a decrease of

striated band size at its ends as shown in Fig. 3C,D. This

indicates a spindle-like shape of many of these structures. We

identified four major morphological types of localization of

leptofibrils in the infarcted cardiac muscle: (1) their attachment

to nascent, resorbing or assembled myofibrils (Figs. 1A–D,

4A); (2) direct focal or multifocal attachment of leptofibrils to

the sarcolemma through the lateral surfaces of their Z-band

structures without any contacts with myofibrils or intercalated

E.I. Dedkov et al. / Micron 38 (2007) 659–667662

Fig. 1. Formation of clusters of leptofibrils in the areas of developing cytoplasmic processes on the free ends of cardiac myocytes in the regions of resorbed

intercalated discs in the perinecrotic area (A) and (B). Panels C and D are higher magnification images of the areas shown in (A) and (B), respectively, that focus on the

details of morphological interrelations of leptofibrils and other subcellular structures. Arrows indicate direct contacts of leptofibrillar bundles with Z-bands of

partially resorbed and nascent myofibrils. Note that the presence of leptofibrils is topographically associated with dense network of intermediate filaments (asterisks in

C and D) and subsarcolemmal vesicular structures and tubular structures of the sarcoplasmic reticulum (arrowheads in C and D). A and B illustrate two serial sections

through the same area, 10 days after infarction. Scale bars, 1 mm (A and B), 0.5 mm (C and D).

discs (Figs. 2A–C, 3A–D); (3) attachment of leptofibrils to

subsarcolemmal accumulations of electron dense Z-band

material in newly formed fascia adherentes of the remodelled

intercalated discs (Fig. 4A,B); and (4) formation of clusters of

leptofibrils contacting one another predominantly at the levels

of their terminal Z bands (Fig. 1C,D). These four patterns are

based on our observations of leptofibrils sectioned at different

angles and on attempts to reconstruct their structure from their

profiles seen in adjacent serial sections. In some cases, the

morphology of leptofibrils can combine the structural

charactertistics of any two types described above. For example,

leptofibrils located in the areas of the intercalated discs develop

contacts with nascent myofibrils (Fig. 4A) or the sarcolemma

(Fig. 4B).

Taken together, the results of this study allow us to make the

following conclusions: (1) formation of leptofibrils is

associated with structural remodeling and myofibrillogenesis

in cardiac myocytes located in the border zone of myocardial

E.I. Dedkov et al. / Micron 38 (2007) 659–667 663

Fig. 2. Leptofibrils (long arrows) in the subsarcolemmal areas of cardiac

myocytes located in the border zone 10 days (A and B) and 15 days (C) after

left ventricular infarction. The area in the frame in the image (A) is shown at a

higher magnification in (B). Abundant vesicular structures (short arrows in B)

are located near leptofibrils in myofibril-free zones (B). Scale bars, 0.5 mm (A),

0.25 mm (B and C).

infarction; (2) assembly of leptofibrils is not related to atrophic

processes, degeneration and death of myocytes because we did

not find these structures in poorly differentiated, atrophying,

degenerating and moribund cells; and (3) detachment of

myofibrils from the sarcolemma and dissociation of inter-

cellular contacts were frequently required for activation of

leptofibril formation.

4. Discussion

The results of this study show that formation of leptofibrils

in vivo requires destabilization of the differentiated state of

cardiac muscle cells and more or less advanced disassembly of

their contractile structures. Interestingly enough, leptofibrils

were also found in cardiac myocytes of mechanically unloaded

papillary muscle of the cat heart (Tomanek and Cooper, 1980).

Dissection of papillary muscle results in partial resorption of

myofibrils and appears to represent another condition of

induced destabilization of normal structure of the myocardial

tissue. Development of leptofibrils was observed in the mouse

heart treated with adriamycin (Payne, 1982) and in human

skeletal muscle transplanted into nude mice (Wakayama et al.,

1980). Of special interest for our discussion are the data

concerning the presence of leptofibrils in tumors of cardiac

muscle and skeletal muscle origin (Fenoglio et al., 1976, 1977;

Bleisch and Kraus, 1980; Carstens and Martin, 1986; Duyvene

and Wit, 1986; Kim et al., 1989; Tanimoto and Ohtsuki, 1996),

in cell cultures isolated from normal rat and cat heart

(Martynova and Borisov, 1987; Simpson et al., 1993) and in

embryonic human skeletal muscle (Askanas et al., 1978).

Muscle tumors are very heterogeneous cell populations in terms

of levels of differentiation of individual cells. Typical

characteristics of these systems include blocked or incomplete

differentiation of the majority of the cell population, instability of

differentiative properties, and susceptibility to more or less

advanced dedifferentiation. The presence of leptofibrils in the

myocardium of infants suffering from cardiomyopathy (Silver

et al., 1980; Saffitz et al., 1983) appears to represent another

example of the development of these structures under conditions

of incomplete or abnormal differentiation. Small numbers of

leptofibrils were also found in specialized types of muscle cells

with relatively poorly developed contractile function, such as

muscle spindles in skeletal muscle of the frog, fowl, rat and

human (Karlsson and Andersson-Cedergren, 1968; Rumpelt and

Schmalbruch, 1969; Adal, 1977; Low et al., 1983) and the

conductive system of the heart (Caesar et al., 1958; Forbes and

Sperelakis, 1980; Forsgren and Thornell, 1981; Thornell and

Eriksson, 1981). Very similar if not identical structures were also

described in denervated and injured somatic muscle in the crop of

the cockroach Leucophaea maderae (Taylor, 1967). The

presence of leptofibrils in muscle cells of animals that belong

to taxonomically diverse systematic groups indicates that the

capacity to assemble these structures is an evolutionary

conserved process.

We have not found any reports concerning the presence of

leptofibrils in rat myocytes at postnatal stages of development

in vivo despite a large number of published studies focused on

E.I. Dedkov et al. / Micron 38 (2007) 659–667664

Fig. 3. Subsarcolemmal leptofibrils associated with the areas of development of intercellular contacts between muscle cells located in the perinecrotic zone. (A) Two

contacting myocytes 7 days following myocardial infarction. Double arrows show the region containing leptofibrils beneath the sarcolemma in the area of contact

between two cells. This region is presented at a higher maginification in the inserted photograph. The long arrow in the insert indicates the localization of leptofibrils,

and the short arrow shows the Z band of a myofibril. Leptofibrils marked with arrowheads in the panel (A) are shown at a higher magnification in panels (B) and (C).

Panel B shows the area near upper arrowhead at a high magnification. The small arrows in panel B indicate subsarcolemmal bodies of Z band-like material located in

both cells at the same level and organized in the periodic pattern typical of leptofibrils. Note the presence of Z-bands of nascent myofibrils (long arrows in B) and

invaginated caveolae on the sarcolemma localized near leptofibrils. Two serial sections through the same area of contacting cardiac myocytes (C and D) clearly show

the association of leptofibrils (arrowheads) with Z-bands of normal myofibrils (arrows). Scale bars, 0.5 mm (A, C and D), 0.25 mM (B).

the ultrastructure of these cells. This fact can be considered as

the evidence of their rare occurrence in normal working

terminally differentiated muscle cells of adult animals.

Formation of leptofibrils in the infarcted rat myocardium

appears to be associated with intense remodeling and partial

dedifferentiation of cardiac myocytes and acquisition of some

structural characterisctics typical of embryonic tissue. Taken

together, all these data support our observations that the

formation of leptofibrils follows destabilization of the

differentiated phenotype in muscle cells.

Due to the lack of information concerning the functional

role and morphogenesis of leptofibrils, we will briefly focus

our discussion on this issue. There are several hypotheses

concerning the functions of leptofibrils. The authors who

studied intrafusal muscle fibers noted the close association of

leptofibrils with axonal terminals and related their function to

the involvement in the sensory function of the muscle spindles

(Rumpelt and Schmalbruch, 1969; Adal, 1977). Hypotheses

concerning the functional role of leptofibrils in cardiac muscle

cells of different species can be divided into three major

groups: (1) leptofibrils perform a mechanical protective role;

(2) leptofibrils participate in myofibrillogenesis; (3) leptofi-

brils are aberrant structures. The first two viewpoints are

discussed in detail by Bogusch (1978) and Myklebust and

Jensen (1978) and the third possibility is discussed by Walker

et al. (1975).

E.I. Dedkov et al. / Micron 38 (2007) 659–667 665

Fig. 4. Leptofibrils in the areas of nascent fasciae adherentes of left ventricular cardiac myocytes 10 days (A) and 15 days (B) after myocardial infarction. Note

termination of leptofibrils near nascent myofibrils in A and in the subsarcolemmal Z-band material in the area of the intercalated disc in B (long arrows). Short arrows

in B show Z-band materialin the raea of a developing fascia adherentia. Note the absence of organized actin filaments typically located in this area. Arrowheads

indicate vesicular structures typically associated with leptofibrils. Scale bars, 0.5 mm (A) and 0.25 mm (B).

The view on the role of leptofibrils in myofibrillogenesis

evolved from the initial concept of direct transformation of

leptofibrils into myofibrils and then to consideration of some

organizing role of these structures in assembly of sarcomeres.

Ruska and co-workers, who discovered leptofibrils in growing

skeletal and cardiac muscle (Ruska and Edwards, 1957;

Thoenes and Ruska, 1960) called the newly found structures the

‘‘prodromal pattern’’, or ‘‘leptomeric myofibrils’’. They

considered leptofibrils as direct structural precursors of

myofibrils serving as a scaffold for assembly of sarcomeres

by means of intercalation of actin and myosin filaments

between Z-bands of leptofibrils. This would result in

progressive growth and formation of sarcomeric structures of

the normal size. Although the exact molecular mechanisms of

myofibril assembly are still not completely clear, studies of

myofibrillogenesis performed during the last two decades

apparently do not discuss the involvement and possible role of

leptofibrils in the formation of contractile structures (e.g.

Sanger et al., 2004). In our study, we did not find any evidence

of direct structural transition from leptofibrils to myofibrils and

also did not find any illustrations of such transitional forms in

publications of other authors. Therefore, despite the fact that

formation of leptofibrils is temporally and spatially associated

with activated myofibrillogenesis, there is no experimental

evidence supporting direct transformation of leptofibrils into

myofibrils. However, we should note that most of modern

studies of myofibril assembly are based on the use of

fluorescent probes for localization of individual contractile

proteins in cultured skeletal and cardiac muscle cells (reviewed

by Sanger et al., 2006). Unfortunately, this approach does not

allow one to perform high-resolution spatial ultrastructural

analysis of the dynamics of myofibril assembly which would

include the investigation of the role in this process of such small

structures as leptofibrils and other minor cytoplasmic compo-

nents. There are indications that the protein composition of

myofibrils and leptofibrils appears to be different from the

protein composition of myofibrils (Bogusch, 1976; Hosokawa

et al., 1994). The fact that leptofibrils can be found in the area of

the nascent fascia adherentes at the sites of typical location of

actin filaments near Z-discs (Fig. 4B) and their frequently

observed direct connection with Z-discs (Fig. 1) suggests that

their filaments may interact with a-actinin or other Z-disc

proteins. Nahirney et al. (2006) recently suggested that

leptofibrils may be somehow involved in the dynamic

remodeling of Z-bands under conditions of intense muscle

function in teleost fish skeletal muscle. Our study of the

mammalian heart during post-injury remodeling suggest that

the development of leptofibrils may be associated with stress

related to functional overload. The presence of these structures

in the myofibril-free zones near the nascent sarcomeres may

reflect their involvement in the establishment of proper

structural relationships between the sarcolemma, the nascent

sarcomeres and other cytoskeletal elements.

To understand better a possible functional role of

leptofibrils, we also should briefly discuss some specific

cellular responses of cardiac muscle to ventricular infarction.

The process of compensatory remodeling in the overloaded

heart is accompanied by re-expression of at least several

structural and biochemical markers typical of the fetal

myocardium (for review see Borisov, 1998). One of the most

important compensatory responses of the overloaded heart is

progressive cellular hypertrophy accompanied by formation of

additional amounts of contractile structures. This process has

been described under different conditions of functional

overload of the heart including myocardial infarction (for

literature see Borisov, 1991; Cox et al., 1991; Wang et al.,

1999). This indicates that activation of myofibrillogenesis and

leptofibrillogenesis may occur and progress at the stage of

compensatory hypertrophic growth of cardiac muscle cells

following myocardial infarcton.

E.I. Dedkov et al. / Micron 38 (2007) 659–667666

Thus, our data do not support the conclusion that leptofibrils

are directly involved in myofibrillogenesis as direct structural

precursors of sarcomeres. At the same time, the results of this

study demonstrate the temporal association of leptofibrillogen-

esis and assembly of myofibrils in cardiomyocytes following

myocardial infarction. The results if this study suggest that

formation of leptofibrils is not related to atrophic and

prenecrotic changes in cardiac muscle cells. Therefore,

leptofibrils are not aberrant structures because their formation

correlates with an advanced level of differentiation and is not

observed in poorly differentiated and degenerating muscle

cells. On the basis of our data, we can conclude that the

development of leptofibrils requires the presence of mature or

nascent myofibrils and appears to be associated with the process

of cell remodeling and redifferentiation. The authors are

thankful to Dr. B.M. Carlson for support of this work.

Acknowledgments

Supported by NIH (R01 HL075093-1), and the Muscle

Dystrophy Association (MDA3803). E.I.D. was a recipient of a

fellowship from the Department of Higher Education of the

Russian Federation. The authors are thankful to Dr. B.M.

Carlson for support of this work.

References

Adal, M.N., 1977. Leptofibrils in intrafusal muscle fibers of muscle spindles in

the domestic fowl. Cell Tissue Res. 184, 281–286.

Arbustini, E., 1988. Leptofibrils in cardiac myocytes. Ultrastruct. Pathol. 12,

251–254.

Askanas, V., Engel, W.K., Bethelem, J., 1978. Leptomeres in cultured human

muscle. Acta Neuropathol. 42, 247–250.

Bleisch, V.R., Kraus, F.T., 1980. Polyploid sarcoma of the pulmonary trank:

analysis of the literature and report of a case with leptomeric organelles

and ultrastructural features of rhabdomyosarcoma. Cancer 46, 314–

324.

Bogusch, G., 1975. Electron microscopic investigations on leptomeric fibrils

and leptomeric complexes in the hen and pigeon heart. J. Mol. Cell. Cardiol.

7, 733–745.

Bogusch, G., 1976. Enzymatic digestion and urea extraction on leptomeric

structures and normomeric myofibrils in heart muscle cells. J. Ultrastruct.

Res. 55, 245–256.

Bogusch, G., 1978. Electron microscopic studies on leptomere structures in

Purkinje cells and energy muscle cells of bird hearts. Verhandlungen der

anatomischen Gesellschaft 72, 289–294.

Borisov, A.B., 1991. Myofibrillogenesis and reversible disassembly of myofi-

brils as adaptive reactions of cardiac muscle cells. Acta Physiol. Scand. 142

(suppl 599), 71–80.

Borisov, A.B., 1998. Cellular mechanisms of myocardial regeneration. In:

Ferretti, P., Geraudie, J. (Eds.), Cellular and Molecular Basis of Regenera-

tion. from Invertebrates to Humans. John Wiley & Sons, Chichester,

London, New York, pp. 335–353.

Bugaisky, L., 1991. Cardiac muscle plasticity: myocyte growth and differentia-

tion. In: Oberpriller, J.O., Oberpriller, J.C., Mauro, A. (Eds.), The Devel-

opment and Regenerative Potential of Cardiac Muscle. Harwood Academic

Publishers, New York, pp. 333–349.

Caesar, R., Edwards, G.A., Ruska, H., 1958. Electron microscopy of the

impulse conductive system of the sheep heart. Zeitschrift fur Zellforschung

und mikroskopische Anatomie 48, 698–719.

Carstens, P.H.B., Martin, A.W., 1986. Soft tissue tumor with prominent

leptomeric fibrils and complexes. Ultrastruct. Pathol. 10, 137–144.

Cox, M.M., Berman, I., Myerburg, R.J., Smets, M.J.D., Kozlovskis, P.L., 1991.

Morphometric mapping of regional myocyte diameters after healing of

myocardial infarction in cats. J. Mol. Cell. Cardiol. 23, 127–135.

Duyvene, D.E., Wit, L.J., 1986. Fine cytofilaments in a metastatic juvenile

rhabdomyosarcoma. Ultrastruct. Pathol. 10, 107.

Fenoglio, J.J., Mcallister, H.A., Ferrans, V.J., 1976. Cardiac rhabdomyoma: a

clinicopathological and electron microscopic study. Am. J. Cardiol. 38,

241–251.

Fenoglio, J.J., Diana, D.J., Bowen, T.E., Mcallister, H.A., Ferrans, V.J., 1977.

Ultrastructure of a cardiac rhabdomyoma. Hum. Pathol. 8, 700–706.

Forbes, M.S., Sperelakis, N., 1980. Structures located at the Z bands in mouse

ventricular myocardial cells. Tissue Cell 12, 467–489.

Forsgren, S., Thornell, L.E., 1981. The development of Purkinje fibers and

ordinary myocytes in the bovine fetal heart. An ultrastructural study. Anat.

Embryol. 162, 127–136.

Ghadially, F.N., 1997. Intracytoplasmic banded structures. In: Ghadially, F.N.

(Ed.), Ultrastructural Pathology of the Cell and Matrix. fourth ed. Butter-

worth–Heinemann, Boston, Oxford, pp. 1064–1066.

Hosokawa, T., Okada, T., Kobayashi, T., Hashimoto, K., Seguschi, H., 1994.

Ultrastructural and immunocytochemical study of the leptomeres in the

mouse cardiac muscle fibre. Histol. Histopathol. 9, 85–94.

Hulland, T.J., 1988. Leptomeric fibrils in the myocardial fibers of a foal. Vet.

Pathol. 25, 175–177.

Karlsson, U., Andersson-Cedergren, E., 1968. Small leptomeric organelles in

intrafusal muscle fibers of the frog as revealed by electron microscopy. J.

Ultrastruct. Res. 23, 417–426.

Kim, C.J., Cho, J.H., Chi, J.G., Kim, Y.J., 1989. Multiple rhabdomyoma of the

heart presenting with a congenital supraventricular tachicardia—report of

case with ultrastructural study. J. Korean Med. Sci. 4, 143–147.

Low, W.D., Chew, E.C., Kung, L.S., Hsu, L.C., Leong, J.C., 1983. Ultrastruc-

tures of nerve fibers and muscle spindles in adolescent idiopathic scoliosis.

Clin. Orthop. 174, 217–221.

Martynova, M.G., Borisov, A.B., 1987. Leptofibrils in ventricular cardiomyo-

cytes of rat in vitro. Tsitologia 29, 17–21.

Martynova, M.G., Borisov, A.B., 1990. Ultrastructural differentiation charac-

teristics of rat atrial and ventricular cardiac muscle cells in culture. Acta

Morphol. Hung. 38, 17–26.

Myklebust, R., Jensen, H., 1978. Leptomeric fibrils and T-tubule desmosomes in

the Z-band region of the mouse heart papillary muscle. Cell Tissue Res. 188,

205–215.

Nag, A.C., Lee, M.-L., 1990. Differentail response of cultured adult cardiac

muscle cells to a tumor promotor: analysis of myofibrillar organization.

Tissue Cell 22, 655–672.

Nag, A.C., Lee, M.-L., Sarkar, F.H., 1996. Remodeling of adult cardiac muscle

cells in culture: dynamic process of disorganization and reorganization of

myofibrils. J. Muscle Res. Cell Motil. 17, 313–334.

Nahirney, P.C., Fischman, D.A., Wang, K., 2006. Myosin flares and actin

leptomeres as myofibril assembly/disassembly intermediates in sonic mus-

cle fibers. Cell Tissue Res. 324, 127–138.

Payne, C.M., 1982. A quantitative analysis of leptomeric fibrils in an adria-

mycin/carnitine chronic mouse model. J. Submicrosc. Cytol. 14, 337–

345.

Rumpelt, H.-J., Schmalbruch, H., 1969. Zur Morphologie der Bauelemente von

Musckelspindeln bei Mensch und Ratte. Zeitschrift fur Zellforschung und

mikroskopische Anatomie 102, 601–630.

Rumyantsev, P.P., 1991. Growth and Hyperplasia of Cardiac Muscle Cells.

Harwood Academic Publishers, London, New York.

Ruska, H., Edwards, G.A., 1957. A new cytoplasmic pattern in striated muscle

fibers and its possible reation to growth. Growth 21, 73–88.

Saffitz, J.F., Ferrans, V.J., Rodrigues, E.R., Lewis, F.R., Roberts, W.C., 1983.

Histiocytoid cardiomyopathy: a cause of sudden death in apparently healthy

infants. Am. J. Cardiol. 52, 215–217.

Sage, M.D., Jennings, R.B., 1989. Alterations to subsarcolemmal leptomeres in

adult canine myocytes during total in vitro ischemia. Lab. Invest. 61, 171–

176.

Sanger, J.W., Sanger, J.M., Franzini-Armstrong, C., 2004. Assembly of the

skeletal muscle cell. In: Engel, A.G., Francini-Armstrong, C. (Eds.),

Myology: Basic and Clinical. McGraw–Hill, New York, pp. 45–65.

E.I. Dedkov et al. / Micron 38 (2007) 659–667 667

Sanger, J.W., Kang, S., Siebrands, C.C., Freeman, N., Du, A., Wang, J., Stout,

A.L., Sanger, J.M., 2006. How to build a myofibril. J. Muscle Res. Cell

Motil. Feb. 8, 1–12 (E-pub ahead of print).

Selye, H., Bajusz, E., Grasso, S., Mendell, P., 1960. Simple technique for

the surgical occlusion of coronary vessels in the rat. Angiology 11,

398–407.

Silver, M.M., Burns, J.E., Sithe, R.K., Rowe, R.D., 1980. Oncocytic cardio-

myopathy in an infant with oncocytosis in exocrine and endocrine glands.

Hum. Pathol. 11, 598–600.

Simpson, D.G., Decker, M.L., Clark, W.A., Decker, R.S., 1993. Contractile

activity and cell-cell contact regulate myofibrillar organization in cultured

cardiac myocytes. J. Cell Biol. 123, 323–336.

Sussman, M.A., Baque, S., Uhm, C.S., Daniels, M.P., Price, R.L., Simpson, D.,

Terracio, L., Kedes, L., 1998. Altered expression of tropomodulin in

cardiomyocytes disrupts the sarcomeric structure of myofibrils. Circ.

Res. 82, 94–105.

Tanimoto, T., Ohtsuki, Y., 1996. The pathogenesis of so-called cardiac rhab-

domyoma in swine: a histological, immunohistochemical and ultrastruc-

tural study. Virchows Archiv 427, 213–221.

Taylor, R.L., 1967. A fibrous banded structure in a crop lesion of the cockroach,

Leucophaea maderae. J. Ultrastruct. Res. 19, 130–141.

Thoenes, W., Ruska, H., 1960. Uber leptomere Myoibrillen’’ in der Herzmus-

kelzelle. Zeitschrift fur Zellforschung und Mikroskopische Anatomie 51,

560–570.

Thornell, L.E., Eriksson, A., 1981. Filament systems in the Purkinje fibers of the

heart. Am. J. Physiol. 241, H291–H305.

Tomanek, R.J., Cooper, G., 1980. Morphological changes in the mechanically

unloaded myocardial cell. Anat. Rec. 200, 271–280.

Wakayama, Y., Schotland, D.L., Bonilla, E., 1980. Transplantation of human

skeletal muscle to nude mice: a sequential morphologic study. Neurology

30, 740–748.

Walker, S.M., Schrodt, G.R., Currier, G.J., 1975. Evidence for a structural

relationship between successive parallel tubules in the SR network and

supernumerary striations of Z line material in of the chicken, sheep, dog and

Rhesus monkey heart. J. Morphol. 147, 459–473.

Wang, X., Li, F., Campbell, S.E., Gerdes, A.M., 1999. Chronic pressure

overload cardiac hypertrophy and failure in guines pigs. II. Cytoskeletal

remodelling. J. Mol. Cell. Cardiol. 31, 319–331.