Article

ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 12, pp. 1957-1969 © Pleiades Publishing, Ltd., 2025.

Russian Text © The Author(s), 2025, published in Biokhimiya, 2025, Vol. 90, No. 12, pp. 2063-2076.

1957

Mitochondrial Reticulum in Skeletal Muscles:

Proven and Hypothetical Functions

Lora E. Bakeeva

, Valeriya B. Vays

, Irina M. Vangeli

Chupalav M. Eldarov

1,2

, Vasily A. Popkov

, Ljubava D. Zorova

1,2

Savva D. Zorov

1,3

, and Dmitry B. Zorov

1,2,a

Belozersky Research Institute of Physico-Chemical Biology, Lomonosov Moscow State University,

119991 Moscow, Russia

Kulakov National Medical Research Center of Obstetrics, Gynecology, and Perinatology;

Ministry of Health of the Russian Federation, 117997 Moscow, Russia

Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University,

119991 Moscow, Russia

e-mail: zorov@belozersky.msu.ru

Received July 8, 2025

Revised October 12, 2025

Accepted October 19, 2025

Abstract— The mitochondrial reticulum of skeletal muscles has been characterized in the 1970-80s. It has

been suggested and then proven its role is delivering energy in a form of transmembrane potential on the

mitochondrial inner membrane throughout the cell volume, followed by ATP synthesis by the mitochondrial

ATP synthase. However, the data on the mitochondrial ultrastructure still remains a subject to criticism.

Toexclude the possibility of artifacts caused by the sample preparation for electron microscopy, we compared

the structure of mitochondria in the ultrathin sections of muscle fibers observed by electron microscopy and

in intact fibers stained with a membrane potential-dependent dye and visualized by confocal microscopy.

Thecomparison was carried out for mice and naked mole rats known for their superior longevity. The obtained

results confirmed previous findings regarding the structure of mitochondrial reticulum. A model suggesting

the functioning of giant mitochondria as intracellular structures preventing tissue hypoxia was proposed.

DOI: 10.1134/S000629792560190X

Keywords: mitochondria, ultrastructure, reticulum, membrane potential, hypoxia, oxygen transport, mouse,

naked mole rat

* To whom correspondence should be addressed.

INTRODUCTION

According to the concepts established based on

the chemiosmotic theory by Peter Mitchell, mito-

chondrial energetics is determined by the intrami-

tochondrial coupling of the oxidation of respiratory

substrates and ATP generation [1-3]. The energy gen-

erated by the activity of mitochondrial proton pumps

(complexes I, III, and IV) is stored in two forms: the

difference in the hydrogen ion concentrations on

both sides of the inner mitochondrial membrane

(IMM) (ΔpH; the exterior being more acidic than the

matrix) and the difference in the electrical charges

in these compartments (negative charge of the ma-

trix side). This energy, in particular its electrical form

(ΔΨ), is used for the rotation of a portion of the ATP

synthase complex with the generation of ATP [4, 5].

Later, it was shown that mitochondria also use potas-

sium energetics, in which the transport of potassium

ions through the ATP synthase complex also controls

the synthesis of ATP [6-9]. The main component in

both types of mitochondrial energetics is the mito-

chondrial membrane potential created by the respi-

ratory chain [10].

The maintenance of the optimal balance be-

tween the energy production and expenditure is a

rather significant issue [11], especially under stress

conditions, which requires increased ATP production

BAKEEVA et al.1958

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

in order to adequately supply the entire cell volume

with oxidative substrates and oxygen and/or with ATP

to power endergonic reactions. The primary supplier

of oxygen and nutrient substrates is the circulatory

system that delivers them to tissues. An imbalance

created when the consumption of oxygen and sub-

strates exceeds their delivery to compartments locat-

ed downstream in the circulation system results in

ischemia/hypoxia. When a quick switch to glycolysis

is impossible (even if the efficiency of glycolysis as

an energy-producing mechanism is low), both hypox-

ia and ischemia can have fatal consequences. This

imbalance can be avoided by reducing the depen-

dence on the oxidative energy sources, i.e., by de-

livering ATP evenly throughout the tissue. There are

several possible scenarios providing an adequate ATP

supply to the entire cellular volume. In one of them,

ATP is produced by the mitochondria located near

the source of oxygen and oxidation substrates (blood

capillaries), after which it diffuses into the tissue.

However, this diffusion is limited by the existence

of various intracellular protein and lipid structures

that act as a barrier to ATP diffusion, which prevents

the cells from maintaining a balance between the

energy intake and expenditure within the entire cell

volume, especially under stress conditions.

Another possibility for providing energy to all

cellular compartments can be realized if the cells lo-

cated between the blood capillaries are crossed by

a continuous, gap-free mitochondrial network with a

preferential location of proton pumps at the sourc-

es of substrates and oxygen and with a uniform dis-

tribution throughout the network of ATP synthase

complexes ready to provide ATP synthesis at any

moment. In this case, the network will be energized

by the activity of proton pumps located near the cap-

illaries, while ATP can be produced at any site within

the cell volume due to the uniform distribution of ΔΨ

(equipotentiality) along the entire length of the mito-

chondrial network. This second scenario, was theoret-

ically justified by V.  P.  Skulachev in 1969 for the IMM

and other coupling membranes (e.g., chloroplast and

bacterial ones) and allowed to formulate the theory

of coupling membranes as “electrical cables” used for

the rapid and efficient transfer of electrical energy

in the cell [12]. In particular, it was suggested that

IMMs act as intramitochondrial “electrical wires” for

providing an adequate supply of ATP to the cellular

volume.

It should be admitted that before the develop-

ment of mitochondria visualization methods using

fluorescent probes, the optical limitations of conven-

tional light microscopy together with a rapid devel-

opment of electron microscopy, had led to the loss

of the intuitive perception of the three-dimensional

structure of mitochondria based on two-dimensional

electron microscopy image. A significant progress has

been achieved when the scientists started to use seri-

al ultrathin sections for the three-dimensional recon-

struction of cells and most importantly, the mitochon-

dria. A breakthrough was the use of this approach

for the reconstruction of the mitochondrial network

in the diaphragm (striated) muscle, which revealed

that in rat diaphragm muscle fibers, the bulk of mi-

tochondrial material was located in a plane perpen-

dicular to the long axis of the muscle fiber, in the

isotropic zones on both sides of the Z-line in a form

of layers consisting of extended, branched mitochon-

dria. Accordingly, in each muscle fiber, the number

of such mitochondrial layers was equal to the num-

ber of Z-lines multiplied by two. All these numerous

layers were interconnected and formed a single mi-

tochondrial system represented by the vertical rows

of mitochondria running along the myofibrils. This

structure of the mitochondrial apparatus was named

the mitochondrial reticulum [13]. However, in the di-

aphragm muscles of rat embryos and neonatal rats,

the entire mitochondrial system was represented by

small, single, non-branching, elongated mitochon-

dria located along the myofibrils [14]. In 1978, com-

pelling arguments were obtained in support of the

theory of mitochondria functioning as intracellular

electrical cables. Based on the idea that IMMs act as

mitochondrial electrical cables extending over long

distances without breaks, it has been found that mi-

tochondrial branches contact through the osmiophilic

electron-dense junctions, while the IMMs themselves

do not form physical contacts with each other [13].

Such electron-dense junctions have been found in

abundance in rat cardiomyocytes, suggesting that the

mitochondrial reticulum in cardiac cells is formed by

multiple clusters of mitochondrial branches connect-

ed by the mitochondrial junctions [15].

In 1986-1988, the theory of mitochondria as elec-

trical cables has been experimentally confirmed, first,

for the filamentous mitochondria of fibroblasts and

then for the mitochondria of neonatal cardiomyocytes

formed by establishing the contacts between the mi-

tochondrial clusters [16,  17]. It was shown that local

deenergization of mitochondria led to the depolariza-

tion of the entire mitochondrial cluster, including its

connections formed by the junctions, thus indicating

the possibility of electrical communication between

the IMMs. It was suggested that the junctions can

be in the “on” or “off” state, depending on the need

for a certain size of the equipotential mitochondri-

al cluster. Several decades later, similar conclusions

were made when the three-dimensional organization

of striated muscle cells was assessed with modern

methods, using the concept of mitochondria as ex-

tended power plant [18, 19]. However, despite the ob-

tained evidence, there is still an occasional criticism

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BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

of the methodology employed to assess the mitochon-

drial reticulum organization in skeletal muscles, most-

ly, of procedures used for the sample preparation in

electron microscopy. Hence, this required the use of

other methods, whose results could be compared with

the electron microscopy data. Such arguments have

become the basis for our study, in which we com-

pared the mitochondrial organization in vital skeletal

muscle sections observed by confocal microscopy with

the images obtained by conventional electron micros-

copy. The muscles were isolated from mice and naked

mole rats, as the latter have a superiorly long lifes-

pan associated, in part, with the structural and func-

tional characteristics of their mitochondria [20-23].

MATERIALS AND METHODS

Laboratory animals. Mice. Male 2.5-month-old

C57Bl/6 mice (n  =  5) were housed in individually

ventilated cages (IVCs), with a 12/12 light/dark cycle

at 20-24°C with ad libitum access to food and water.

Safe BK 8/15 wood chips (JRS, Germany) were used

as bedding.

Naked mole rats (Heterocephalus glaber). Two

groups of naked mole rats (6-week- and 7-year-old)

were used in the experiments; each group contained

four animals. Given that the lifespan of a naked mole

rat is ~30 years, a 7-year-old animal is approximately

equivalent to a young mouse assuming that the mouse

lifespan is ~1.5-2 years. Naked mole rats were taken

from the colonies maintained in plexiglass mazes at

the Belozersky Institute of Physico-chemical Biology,

Lomonosov Moscow State University kept at 26–29°C

and relative humidity of 60-80%. Food was ad libitum

and included sweet potatoes, carrots, apples, fennel,

cereals with vitamins and minerals and oatmeal.

Visualization of muscle vital sections by con-

focal microscopy. Quadriceps femoris muscles were

collected from the animals anesthetized with 2.5%

isoflurane using a SomnoSuite® system (Kent Scien-

tific Corporation, USA) and sacrificed by decapitation.

The samples were placed in the incubation medium

(DMEM/F12 without sodium bicarbonate; PanEco,

Russia) to remove blood, after which they were em-

bedded in low-melting-point agarose (Thermo Fischer

Scientific, USA). Sections (70-100 μm thick) were pre-

pared with a Leica VT-1200s vibratome (Leica Bio-

systems, UK), washed with the incubation medium,

and incubated for 30  min with 200  nM tetramethyl-

rhodamine ethyl ester (TMRE), Thermo Fisher Scien-

tific). All procedures were performed at 25°C. Mito-

chondria in the TMRE-loaded muscle sections were

visualized using an LSM 710 inverted laser confocal

microscope (Carl Zeiss, Germany) with excitation at

543  nm and emission >560  nm.

Electron microscopy. Excised muscle tissue

samples were fixed with 3% glutaraldehyde (Sigma-

Aldrich, USA) in 0.1  M phosphate buffer (pH  7.4) for

2  h at 4°C and then with 1% osmium tetroxide for

1.5  h, dehydrated in a series of increasing ethanol

concentrations of 50, 60, 70, 80, and 96% (70% etha-

nol contained 1.4% uranyl acetate (Serva, Germany)

to enhance the contrast) and embedded in Epon812

epoxy resin. A series of sequential ultrathin sections

were prepared using a Leica ultramicrotome (Leica

Biosystems). Visualization was performed with a

JEM1400 electron microscope (JEOL, Japan) equipped

with a QUEMESA camera (Olympus, USA) at an accel-

erating voltage of 100  kV and beam current of 65  μA.

The images were processed using the software pro-

vided by the manufacturer (EMSIS GmbH, Germany).

RESULTS

Figure 1a shows an image taken from the skeletal

muscle fiber (m.  quadriceps) section from a 3-month-

old mouse obtained by confocal microscopy. The sec-

tions were treated for 20-30 min with the ΔΨ-depen-

dent probe TMRE for 20-30min immediately after the

animal had been sacrificed, which made possible the

detection of energized mitochondria due to the differ-

ence in the mitochondrial fluorescence intensity rela-

tive to the cytoplasm. The use of the fluorescent dye

allowed to observe in real-time both the morphology

of the mitochondrial apparatus (all cellular structures

that fluoresced were mitochondria) and the level of

mitochondrial activity (the higher the TMRE fluores-

cence intensity, the higher the mitochondrial energi-

zation). The entire cross-sectional area of the muscle

fiber was packed with a dense network of branched,

energized mitochondria. The obtained image of the

mitochondrial system structure in live skeletal mus-

cle fibers fully corresponded to the ultrastructure of

the mitochondrial reticulum revealed by transmission

electron microscopy (Fig. 1b).

Using the same approach, we analyzed the struc-

ture of the mitochondrial apparatus in the skeletal

muscles (m.  quadriceps) from naked mole rats aged

6 months and 7 years. Figure  2a shows that unlike

the muscle fibers of mice, the muscle cells of mole

rats lacked the mitochondrial network, and their mi-

tochondria are distributed rather randomly.

These data are fully consistent with the assump-

tion that the mitochondrial reticulum is absent in

skeletal muscle fiber of naked mole rat. Even by the

age of 7-11 years, when the organization of the mi-

tochondrial apparatus is permanently established,

it differs from that in mice [23]. It should be not-

ed once again that the lifespan of naked mole rats

reaches 30 years, and an animal at the age of 7-11

BAKEEVA et al.1960

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Fig. 1. Comparison of the mitochondrial apparatus in the skeletal muscle fiber (m. quadriceps) of a 2.5-month-old mouse

revealed by confocal microscopy and transmission electron microscopy: a)confocal microscopy of a TMRE-loaded vital sec-

tion of skeletal muscle fiber; b)electron microscopic image of a fixed section through the muscle fiber. The mitochondrial

reticulum in panel b (dark profiles; arrows indicate mitochondrial elements of the reticulum) forms a single mitochondrial

network organized by the branched mitochondria occupying the entire sectional area of the muscle fiber, similar to that

revealed by confocal microscopy. Both images clearly demonstrate a developed network of the mitochondrial reticulum

formed by a system of thread-like extended mitochondria located in the isotropic region of the muscle fiber.

is approximately equivalent to a sexually mature

mouse, whose lifespan is ~1.5-2 years.

Therefore, the use of intact tissue and its examina-

tion immediately after sampling (i.e., without fixation,

dehydration, or freezing), allowed us to observe the

actual structure of the skeletal muscle mitochondrial

apparatus in a form of reticular (continuous) and non-

reticular (discontinuous) mitochondrial structures.

To obtain more a convincing and unambiguous

evidence of the absence of mitochondrial reticulum

in the skeletal muscles of naked mole rats, we an-

alyzed the mitochondrial profiles in a series of six

sequential ultrathin sections observed by electron mi-

croscopy (Fig.  3). Unlike in mice and rats, the mito-

chondrial structures in naked mole-rats did not form

a single network (reticulum), but were represented by

fragments that did not contact each other. This char-

acteristic mitochondrial organization was observed in

the muscle cells of both young (6-month-old) (Fig.  4)

and mature (7-year-old) animals.

DISCUSSION

The presence of a branched and morphologically

homogeneous mitochondrial network in rat striated

MITOCHONDRIAL RETICULUM IN SKELETAL MUSCLES 1961

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Fig. 2. The structure of mitochondrial population in the skeletal muscle (m.  quadriceps) of a 7-year-old naked mole rat:

a) confocal microscopy image of the muscle fiber vital cross-section showing the absence of mitochondrial network and

a chaotic distribution of individual mitochondria; b) transmission electron microscopy image of the cross-section through

the muscle fiber isotropic zone showing small single mitochondria (dark profiles indicated by arrows); c) enlarged image

of the fixed muscle fiber cross-section.

muscle was first described in 1978 [13] based on

analysis of electron microscopy images. However,

sample preparation for electron microscopy includes

fixation, staining, and dehydration steps, so there is

always a concern that these procedures may alter the

actual morphology existing in an intact tissue. There-

fore, it is advisable to confirm the electron microsco-

py data using other methods excluding the influence