European Journal of Applied Physiology

https://doi.org/10.1007/s00421-021-04863-6

INVITED REVIEW

A century of exercise physiology: key concepts in muscle cell volume

regulation
Michael I. Lindinger1 

Received: 4 August 2021 / Accepted: 27 November 2021

© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2021

Abstract
Skeletal muscle cells can both gain and lose volume during periods of exercise and rest. Muscle cells do not behave as
perfect osmometers because the cell volume changes are less than predicted from the change in extracellular osmolality.
Therefore, there are mechanisms involved in regulating cell volume, and they are different for regulatory volume decreases
and regulatory volume increases. Also, after an initial rapid change in cell volume, there is a gradual and partial recovery of
cell volume that is effected by ion and water transport mechanisms. The mechanisms have been studied in non-contracting
muscle cells, but remain to be fully elucidated in contracting muscle. Changes in muscle cell volume are known to affect the
strength of contractile activity as well as anabolic/catabolic signaling, perhaps indicating that cell volume should be a regu-
lated variable in skeletal muscle cells. Muscles contracting at moderate to high intensity gain intracellular volume because of
increased intracellular osmolality. Concurrent increases in interstitial (extracellular) muscle volume occur from an increase
in osmotically active molecules and increased vascular filtration pressure. At the same time, non-contracting muscles lose
cell volume because of increased extracellular (blood) osmolality. This review provides the physiological foundations and
highlights key concepts that underpin our current understanding of volume regulatory processes in skeletal muscle, begin-
ning with consideration of osmosis more than 200 years ago and continuing through to the process of regulatory volume
decrease and regulatory volume increase.

Keywords  Skeletal muscle · Regulatory volume increase · Regulatory volume decrease · Muscle ion transport · History of
exercise

Abbreviations Pi Inorganic phosphate

ADP Adenosine diphosphate PCr Phosphocreatine
ATP Adenosine triphosphate RVD Regulatory volume decrease
ATPase Adenosine triphosphatase RVI Regulatory volume increase
Ca2+ Calcium cation T system Transverse tubule system
Cl− Chloride anion TRPV2 Transient receptor potential protein V2
K+ Potassium cation
MCT Monocarboxylate transport protein
Mg2+ Magnesium cation Introduction
Na+ Sodium cation
NaCl Sodium chloride The present paper is the first review dealing with vol-
NKA Sodium, potassium ATPase ume regulation in skeletal muscle cells, although an early
NKCC Sodium, potassium, two chloride cotransporter understanding of regulated volume increase in muscle
cells has appeared (Gosmanov et al. 2003a). It is perhaps
timely as it has been more than 120 years since Overton’s
Communicated by Philip D. Chilibeck.
foundational studies of osmotically induced changes in
* Michael I. Lindinger muscle volume, and even longer (three centuries) since
mi.lindinger@gmail.com the development of the first key concepts that underpin our
current understanding of muscle cell volume regulation.
1
The Nutraceutical Alliance Inc., Burlington, ON L7N 2Z9, Regulation (Taylor et al. 2021) implies that cell volume or
Canada

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Vol.:(0123456789)

perhaps osmolality (Zanou et al. 2015) is a physiologically
sensed variable and that control mechanisms are activated
to prevent excessive increases/decreases in cell volume,
i.e. skeletal muscle cells do not behave as perfect osmom-
eters (Urazaev et al. 1987; Sitdikov et al. 1989; Lindinger
et al. 2011).
The foundations of modern exercise physiology were
laid down in the 1920s and 1930s by two main groups of
researchers (David Barr, Harold Himwich et al. at Cornell
University; Bruce Dill, Lawrence J. Henderson et al. at Har-
vard University), with additional important research contri-
butions by people like John Scott Haldane, Claude Gordon
Douglas, August Krogh, Johannes Lindhard, Archibald Viv-
ian Hill, Otto Meyerhof, Francis Benedict, Edward Cathcart,
Francis Bainbridge (Lindinger and Ward 2022). Since that
time, compared to the emphases on muscle biochemistry,
cardiovascular function and respiratory function, there has
been very little research directed at changes in muscle vol-
ume in general, and at the regulation of muscle cell volume
specifically. There are notable reviews that consider various
aspects of volume changes in whole muscle, whether it be
in vivo (Harrison 1985; Sejersted and Sjogaard 2000) or
in vitro (Fenn 1936; Sejersted and Sjogaard 2000) but until
the latter review it was widely believed, incorrectly, that
skeletal muscle cells respond passively to imposed changes
in osmotic strength and essentially behave as near-perfect
osmometers. The one exception is some work by Russian
muscle physiologists in the late 1980s (Urazaev et al. 1987;
Sitdikov et al. 1989) reporting regulatory volume increase in
skeletal muscle cells exposed to solutions of raised osmotic
strength. Notably, however, these reviews point to a seminal
body of research demonstrating the complexity of fluid and
electrolyte movements in muscles and the whole body dur-
ing exercise and recovery, as well as with dehydration and
rehydration. Complex because (1) all cells in the body are
likely involved, (2) volume changes in body fluid compart-
ments are caused by contracting muscles as well as increased
blood pressure and flow, (3) non-contracting muscle and
other tissues (i.e. blood cells) respond differently than con-
tracting muscle cells and (4) prolonged exercise needs to
consider water losses by cutaneous, renal and respiratory
routes. The topics are beyond the focus of the current review.
The focus of this review is on the regulatory responses
that occur in skeletal muscle cells in response to the osmotic
changes that occur with exercise at different intensities.
Emphasis is given to those concepts considered by the
author to be key in the development of current state-of-the-
art of skeletal muscle cell volume regulation, irrespective
of when the concept was first developed. The review there-
fore does not deal with compartmental shifts in fluid which
are well described by Harrison (1985). Also, the informed
reader may hold that there are other key concepts that have
not been adequately considered or perhaps missed entirely.
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European Journal of Applied Physiology
The absence of detailed consideration of a concept, however,
reflects a paucity of experimental data.
It is well known that an increase in cell volume occurs
in active muscles at the onset of contractile activity (Fenn
and Cobb 1936) and that this increase in volume is osmoti-
cally driven (Hamilton et al. 1993; Watson et al. 1993). It
is known that both the interstitial and intracellular volumes
increase in contracting muscle (Sjogaard and Saltin 1982;
Sjogaard et al. 1985). We also know from indirect arterial
and venous blood measures of protein concentration and/or
hematocrit that continued contractile activity at submaxi-
mal intensities is accompanied by a gradual loss of the vol-
ume that was initially gained by contracting muscle cells
(Sjogaard et al. 1985; Sejersted et al. 1986; Lindinger et al.
1994). Cessation of activity results in a further decrease of
interstitial and intracellular muscle volume, and normaliza-
tion of cell volume occurs via several mechanisms includ-
ing metabolic reduction in osmotically active molecules
and transmembrane ion/water transport (Heigenhauser and
Lindinger 1988; Lindinger and Heigenhauser 1988).
What is less known, however, is that non-contracting
muscle in ‘resting’ parts of the body also undergo changes
in cell volume during periods of moderate- to high-intensity
contractile activity (Kowalchuk et al. 1988a; Lindinger et al.
1990). The net movement of water and ions into contract-
ing muscle during exercise results in a decrease in blood
volume and an increase in blood osmolality. The increased
blood osmolality causes non-contracting muscles, and other
tissues, to lose volume when they are perfused with this
blood (Harrison 1985). A quantitative summary of the net
water movements in the body, focusing on contracting and
non-contracting muscles is provided in Fig. 1 using a short-
duration (~ 10 min) incremental exercise test to exhaustion
or short-duration moderate- to high-intensity exercise as
working examples. The 10-min or shorter time frame is
chosen to avoid net losses due to sweating in the calculation
and is based on changes in plasma protein concentration, and
not hemoglobin concentration, because of splenic addition
of erythrocytes at high exercise intensities (Shephard 2016;
Holmstrom et al. 2021).
Regulation of muscle cell volume is important because
volume, or at least intracellular osmolality, appears to be a
‘sensed’ (Zanou et al. 2015) variable. When a physiological
variable is sensed this typically indicates that this variable is
one that can be regulated within the body or even by the cell
itself, as opposed to being controlled (Taylor et al. 2021).
Indeed, when changes in muscle cell volume are imposed
experimentally, there occur seemingly direct changes in
skeletal muscle force development (Gulati and Babu 1982;
Bressler and Matsuba 1991; Rapp et al. 1998), metabolism
(Low et al. 1997; Rennie et al. 1998; Cermak et al. 2009), as
well as whole body glucose homeostasis (Lang et al. 2009;
Hallows et al. 2010) and blood pressure regulation (Kahle
European Journal of Applied Physiology
Fig. 1  Conceptual flow diagram showing main causes of net water
movements during leg exercise, using as an example values that
would be expected at the end of a 10-min incremental exercise test
to exhaustion. Contractile activity in the leg muscles results in an
increase in intracellular osmolytes that, based on measured increases
in plasma protein concentration, results in the net movement of
200 mL of water into contracting muscles (cells and interstitial). The
movement of water from blood, together with the net movement of
osmolytes from contracting muscle into venous blood, raises femo-
ral venous osmolality. Venous blood osmolality is reduced at the right
et al. 2010). Cell volume loss in general, and in muscle spe-
cifically, is associated with an increased metabolism and
catabolism, whereas increased volume is associated with
anabolic effects (Lang 2007; Olsen et al. 2019).
Many of the concepts described in the context of skel-
etal muscle cell volume regulation will be familiar to those
knowledgeable in the areas of epithelial cell, red blood cell,
smooth muscle cell, and cardiac muscle cell volume regula-
tion. Many of the physiological mechanisms first discov-
ered and elucidated in epithelial cells, the most extensively
studied of the aforementioned cell types, were subsequently
found to be similar in skeletal, smooth and cardiac muscle
cells. Despite comprising approximately 40% of the lean
body mass within an individual, however, direct investi-
gation of the mechanisms surrounding volume regulation
within skeletal muscle cells remains sparse. There is increas-
ing evidence that skeletal muscle cells regulate their cell vol-
ume, and do not merely respond passively to impose changes
in intracellular and extracellular osmolality.
While the primary force responsible for volume changes
within any cell is osmotic (Overton 1899; Lang 2007), the
physiological mechanisms that allow for, let alone regulate,
heart as mixed venous from other parts of the body combine with
femoral venous blood. Arterial osmolality is raised above resting and
remains lower than femoral venous. The raised arterial osmolality
results in a net movement of water (up to 100 mL) from non-contract-
ing tissues into the venous circulation—this is calculated from the
difference in plasma protein concentration between arterial blood and
antecubital venous blood (Lindinger et  al. 1992). Additional details
for contracting muscle and non-contracting muscle follow later, and
are summarized in Figs. 4 and 7, respectively
volume changes are complex. Cellular metabolism, the
plasma membrane, water and ion transport systems, an
elaborate cytoskeleton that is contiguous with contractile
proteins, signal transduction systems, intracellular organelles
and domains, as well as extracellular matrix proteins and
neighboring cells are all involved in this well-orchestrated
process (Fig. 2). There are also numerous endocrine and
exocrine signals associated with exercise, some of which are
known to be involved in muscle cell responses, for example
activation of the NKA (Clausen 2003) and the NKCC (Gos-
manov and Thomason 2002; Gosmanov et al. 2002). The
sarcolemma, through its structural linkages with intracellular
proteins (including the contractile apparatus) and extracel-
lular matrix proteins is also a sensor of changes in internal
as well as neighboring cell shape and of molecular informa-
tion—the sarcolemma is well-endowed with receptors on
both the extracellular and intracellular surfaces (Olsen et al.
2019). Some of these receptors (Zanou et al. 2015) must be
associated with ion and metabolite transport proteins (Sejer-
sted and Sjogaard 2000; Gosmanov et al. 2003b), and with
sub-sarcolemmal domains that provide the machinery and
the energy required for transmembrane transport processes,
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Fig. 2  Several of the ion transport systems involved in the physiologi-
cal mechanisms regulating intracellular volume. These components,
as well as aquaporin, are also present in the transverse tubule system.
The subsarcolemmal domain (“fuzzy space”) is shown by the shaded
region interior to the sarcolemma. Cytoskeletal proteins are not
shown for clarity. ­Na+ indicates voltage-sensitive sodium channels;
i.e. the NKA (Semb and Sejersted 1996) and ATP-sensitive
­K+ channels (Weiss and Lamp 1987; Gosmanov et al. 2004).
To summarize, the purpose of this article is to highlight
the key physiological concepts that have led to our current
understanding of volume regulation in skeletal muscle cells
(Fig. 3). In addition to the key concepts within the current
publication, many other important auxiliary concepts (i.e.,
associated physiological structures and processes) are indi-
cated. The narrative essentially begins with Overton’s work
in the late nineteenth century (Overton 1895, 1896, 1899),
with some reference to earlier works, and progresses through
to the present. The interested reader is referred to the follow-
ing reviews to explore some of these key concepts in greater
detail than can be accomplished here (Robinson 1960; Dick
1966; Kleinzeller 1999; Lang 2007; Hoffmann et al. 2009;
Lombard 2014; Brown 2017; Hille 2018; Olsen et al. 2019).
Key concept one: osmosis
Dick referred to osmosis as “by far the most important”
process for water transfer into and out of living cells
(Dick 1966). This necessitates that the cell membrane is
semipermeable, having permeability to water and partial
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European Journal of Applied Physiology
­H2O indicates aquaporins; C­ a2+ ­Mg2+ indicates calcium and magne-
sium channels; ­K+ indicates at least 4 types of potassium channels;
NKA indicates the sodium potassium ATPase; NKCC indicates the
sodium–potassium-chloride-chloride cotransporter. Two examples of
G proteins are indicated, one with the N­ a+ channel and one with the
NKA
impermeability to solutes. The initial description of osmo-
sis has been attributed to Hewson, who recognized that red
blood cells shrank or swelled depending on the salt concen-
tration of the medium—the water ‘flowed’ from a solution
of low salt concentration to one of higher salt concentration
(Hewson 1773). While this finding was largely neglected at
the time, it was rediscovered by Dutrochet who, after per-
forming hundreds of experiments using different solutions
and cell types (membranes), was able to provide a convinc-
ing physical description of water and salt movement, and
the physiological relevance of this phenomenon (Dutrochet
1826). Dutrochet concluded that water moved from the
compartment where the solution was less dense, acidic, or
positively charged to the compartment of greater density,
alkalinity, or negative charge. The concept of an interac-
tion between the intracellular and extracellular environment
explored by Dutrochet, was influenced by previous work by
Porret, who showed that an otherwise ‘impermeable’ animal
membrane could become water-permeable when an electric
current was applied (Porret 1816).
Charles Ernest Overton reported the results of his living
animal and plant cell permeability studies (osmotic proper-
ties”) in a series of five papers between 1895 and 1902. Most
of these were summarized in a lecture delivered to the Natural
European Journal of Applied Physiology
Sciences Research Council in Zurich entitled “About the gen-
eral osmotic properties of the cell, its probable causes and its
significance for physiology” (Overton 1899). In this particu-
lar lecture, Overton makes reference to two papers (Overton
1895, 1896) and more than 10,000 experiments conducted
over a nine-year period. These experiments were a detailed
study of more than 500 small molecules with anesthetic or
toxic properties and the associated osmotic properties of plant
and animal cells. Importantly, these experiments provided a
definitive step toward understanding the cellular physiology
of water permeability.
Key concept two: cell membrane (plasma
skin)
Overton built on the work of many of his predecessors and
contemporaries, as cited in his lectures and publications.
Importantly it was already appreciated that individual cells
were surrounded by a ‘boundary layer’ (cell wall of plants;
epimysium of muscle) as well as a ‘membrane’ overlying the
‘protoplasm’ (Overton 1899). Overton specifically mentions
both in slightly different contexts, although the original texts
are in German. There was controversy at the time, and continu-
ing through the next century, regarding whether cells required
or even possessed a plasma membrane (Pollack 2003; Ling
2004). Cell membranes were first visualized using the elec-
tron microscope (Jones and Barer 1948; Edwards et al. 1956),
though some disputed that the appearance of a membrane was
an artifact of the fixation process. On page 111 of his 1899
publication, Overton states that “the impregnating substances
of the boundary layers of the protoplasts … possess… imme-
diately a “plasma skin” with the same osmotic properties as
the natural boundary layers of the protoplasts.” The quota-
tions and bold font on plasma skin are also found within his
original publication. This followed from page 109, where he
stated “it's become very likely to me that the general osmotic
properties of the cell are due to the fact that the boundary
layers of the protoplast are impregnated with a substance,”
and that the boundary layer is impregnated with “cholesterol
or a cholester … or a mixture of such compounds.” (Overton
1899). This was previously suggested by the earlier research of
Hoppe-Seyler (1866) and Schulze and Likiernik (1891), indi-
cating the composition of the lipids now known to be present
in cellular plasma membranes. This led to the suggestion that
cholesterol and phospholipids could be the main components
of cell membranes (Overton 1899).
Key concept three: selective permeability
(selective solubility)
The concept of a “plasma skin” (cell membrane) is not a
requirement to understanding Overton’s work so long as one
can appreciate that the boundary layer subserves the physi-
ological functions of one-way molecule transfer; ‘selective
solubility’ as he described it. Both one-way molecule trans-
fer (net unidirectional flux) and selective solubility of a mol-
ecule through the boundary layer (selective permeability)
are key concepts in our current understanding of cellular
membrane physiology and cell volume regulation.
With regards to ‘selective solubility’, Overton (1899)
states on page 106: “The osmotic behaviour of the living
protoplast has been compared with that of a precipitation
membrane, yes the boundary layers of the protoplast as a
precipitation membrane, a hypothesis which I also have for
a long time been approaching. For about three years … I
am led to the presumption that the peculiar osmotic prop-
erties of living protoplasts have the appearance of “selec-
tive solubility”, a presumption, which over time almost
led me to the certainty of it.” The quotation marks and
bold font are Overton’s. Foundational work on the permea-
bility of the membrane to sodium chloride (Schmidt 1851;
Bernard 1865; Pfeffer 1877) subsequently led Overton to
state: “Over a period of seventy or more years, it was noted
that when the muscle fibers and other tissue cells from
the sodium chloride-rich blood plasma or lymph should
be rinsed, [there was] not even an approximate balance
between the concentrations of the sodium or chloride ions
in the blood plasma or in [the protoplasmic] fluid of the
tissue cells … so long they are still alive.”
In the 1890s, two theories were presented to explain how
semipermeable membranes operated. First, the pore theory
indicated that membranes had small pores that allowed
them to behave like sieves (Traube 1867). Next, it was theo-
rized that permeating substances actually dissolved into or
through membranes (Nernst 1890). We now appreciate that
both theories hold true for different molecules, although
Overton favored Nernst’s theory based on his extensive
series of experiments (Overton 1895, 1896, 1899).
There is presently a vast body of evidence for the physi-
cal and functional presence of a cell membrane that sepa-
rates the intracellular and extracellular environments. This
membrane is selectively permeable to molecules, allowing
for bidirectional communication between intracellular and
extracellular environments by way of transmembrane pro-
tein linkages and cell signaling molecules (i.e. cytokines).
Permeability to gases, on the other hand, appears to be
largely passive, and the plasma membrane does not hinder
the movement of oxygen or carbon dioxide down their
concentrations gradients (Poole et al. 2022).
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 European Journal of Applied Physiology
◂Fig. 3  A chronological summary of the discovery/development of key
concepts in skeletal muscle cell volume regulation. While aquaporins
must be involved, the nature of their involvement in regulated volume
responses in skeletal muscle cells have yet to be elucidated
Key concept four: unidirectional flux
(one‑way permeation) of water
It was generally understood that all molecules ‘diffused’
across the ‘boundary layer’, and in his many experiments,
Overton demonstrated that small non-polar molecules dif-
fused faster while polar molecules (ions) diffused more
slowly or not all. When the extracellular concentrations of
these molecules (i.e., NaCl) was increased, the cells lost
volume, and vice versa. As a result of these observations,
Overton stated that cells “have the peculiarity that they
are only permeable for water molecules in one direction,
namely from inside to outside,” and believed that some-
thing within the ‘boundary layer’ allowed the movement
of “water molecules probably in the one, but not in the
opposite set direction” (Overton 1899, page 117).
Interestingly, these early observations do not appear to
have inspired the concept of how small molecules may be
involved in the regulation of cell volume. They are, how-
ever, considered foundational to Peter Agre’s discovery
of gated water channels (aquaporins) within the plasma
membranes of erythrocytes and renal tubule epithelial
cells (Denker et al. 1988). This discovery later led to the
2003 Nobel Prize in chemistry, which Agre shared with
Roderick MacKinnon for the latter’s seminal studies on
the structure and operational mechanisms of ion channels.
Key concept five: active ‘diffusion’—the
concept of active transport
Early work with red blood cells (Schmidt 1851) and mus-
cle cells (Bernard 1865) identified that intracellular con-
centrations of ­K+ were much greater than in plasma, while
intracellular ­Na+ and C
­ l− concentrations were opposingly
lower than in plasma. Overton (1899) reported that, in
some cases, there is “an active activity of the protoplasm”
that is involved in maintaining or achieving osmotic pres-
sure by allowing the active transport of molecules into
the cell (page 122). It was already recognized by Overton
that “the intake of potassium into muscle cells is not at all
a purely diosmotic process and that, on the contrary, the
entry is caused by a particular activity of muscle cells”
(Overton 1896). He also stated that potassium entry must
occur with the simultaneous transfer of anions or exchange
with another cation from the muscle cell, otherwise sig-
nificant electrical charge would occur.
Heidenhain synchronously determined that membrane
transport of molecules could not be explained purely by
the physical processes of filtration and diffusion, and intro-
duced the term ‘pump’ to describe the ‘driving force’ for
membrane transport (Heidenhain 1894). Overton (1899)
further elaborated that the energy that powered this driving
force came from the cellular protoplasm. These concepts
are certainly in agreement with our current understanding
of the usage of ATP within the sodium–potassium pump
for potassium influx and sodium efflux (below).
In 1902, Overton reported that contracting muscle gains
“weight” (volume) and NaCl, i.e. “impoverishes the imme-
diate extracellular fluid NaCl concentration, and when
contraction is stopped, then loses NaCl into the immedi-
ate extracellular fluid” (Overton 1902). It was apparent to
Overton that there was an exchange of N ­ a+ and K
­ + ions
across the muscle membrane that was associated with the
change in electrical charge upon muscle contraction. Over-
ton reported that ­Na+ and ­K+ concentrations across the
muscle membrane never equalize in living cells, thus con-
cluding that there needed to be a “mechanism” that main-
tained unequal extra- and intracellular ion concentrations.
This idea remained preliminary in Bernstein’s “membrane
hypothesis” (Bernstein 1902), and remained to be re-dis-
covered and verified by Hodgkin and Huxley (Hodgkin
and Huxley 1945, 1952). Membrane semi-permeability to
sodium was confirmed by Boyle and Conway (Conway
and Boyle 1939), and the exchange of ­Na + for ­K + was
confirmed by Fenn and Cobb (1936), Fenn et al. (1938).
The early understanding of transmembrane ion movement
was largely limited to explanations of passive ion permea-
tion. Even Fenn and Cobb (1934) stated that “potassium
diffuses in and out of muscles by exchange with hydrogen
ions or some equivalent process. Otherwise, it is hard to
explain how potassium can move against the concentration
gradient after a slight increase in the concentration of potas-
sium outside.” Hodgkin and Huxley (1945) stated “Accord-
ing to the conventional membrane theory, the nerve surface
becomes freely permeable to all ions during activity and can
so contribute nothing to the recorded [potential difference].”
These authors did not cite the previous work of Overton or
Bernstein and did not develop the concept of active trans-
port of potassium across the muscle cell membrane. Their
work, however, contributed directly to the body of knowl-
edge relied on by Jens Skou when searching for the source
of energy responsible for active transport.
Skou’s work on nerve fibers identified the energy-requir-
ing process for active ­Na+ extrusion from excitable cells as
a ­Na+/K+-ATPase (NKA) or sodium–potassium pump (Skou
1957). Interestingly, however neither Skou nor others prior
speculated as to a putative location of the sodium–potassium
pump within the cell. It took a further 40 years to dispel
doubts that the sodium–potassium pump was completely
13

responsible for N­ a+/K+ exchange and for Skou to receive
the Nobel Prize in Chemistry in 1997 (Clausen and Persson
1998). Overall, Skou’s work led to the general acceptance
that “water flow is dependent upon active sodium transport”
(Clarkson 1967); a concept that was also further applied to
non-epithelial cell membranes.
Key concept six: molecules move
through protein ‘channels’ or ‘transporters’
situated in cell membranes
The ambiguity of the term “permeability” as applied to bio-
logical membranes was recognized by Teorell (1949), and
in this review, he made clear “the distinction between (a) the
"permittivity" of a membrane for the particular substance
under investigation (i.e., the permeability in the proper,
narrow sense) and (b) the driving forces, which create the
very process of the substance transport (the permeation or
penetration).” He went on to clearly state our current under-
standing: “transport must arise from the simultaneous opera-
tion of both the factors (a) and (b); the presence of either
of them does not alone lead to any penetration across the
membrane.” To further describe transport, he defined the
term “flux” “as the amount of substance which per unit time
penetrates per unit area (of an infinitesimal volume element)
normal to the direction of transport.” This definition con-
formed with Nernst and Planck's fundamental expression for
electrolyte transfer: flux = mobility × concentration × total
driving force. He also recognized that the energy necessary
for substance transport across a biological membrane need
not be “supplied from within this membrane” and that “the
protoplasm may in many instances readily supply the energy
which creates the membrane gradients;” a view held by
Overton (1899) that appears to have been generally accepted
by 1949. It is noteworthy that Teorell did not mention the
word “channel” in his 1949 review, and did not speculate as
to the putative mechanisms for molecular transport.
The idea that molecules are transported through biologi-
cal membranes via “pores” or “channels” predated Teorell
by at least 100 years, and appear to be terms introduced,
or at least used, by von Brücke (1843). The terms pore and
channel were used extensively in research related to permea-
tion through dialysis membranes. For example Weech and
Michaelis (1928) stated: “We thought it probable that the
great difference in the diffusion rates between molecules of
different sizes was due to the fact that the membranes con-
tained pore channels of varying sizes, that only the largest
of these channels would serve for the passage of a large mol-
ecule such as glucose, and that many more channels could be
used by the smaller molecules.” Despite the usage of these
terms, this concept was not generally applied to biological
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European Journal of Applied Physiology
membranes until the late 1950s and early 1960s, particularly
by Hille (1967).
The concept of ion channels and transporters that were
constituent elements within cells membranes was generally
accepted by 1966, with Grundfest summarizing the state-of-
the-art “we can, nevertheless, regard the excitable membrane
as a matrix in which are embedded elements of different
chemical structures, some of the latter forming channels
that are permselective for the different ionic species that
are present in the system” (Grundfest 1966). However, ion
channels, although known to have specificity, were consid-
ered “physical pores formed by faults in the lipid bilayers
of the membrane complex” (Grundfest 1966). Such “pores”
were also considered to be aqueous channels, such that water
could move with ions according to a “friction theory” (Spie-
gler 1958). Such explanations, however, cannot account for
ion channel specificity nor for aquaporins as distinct from
ion channels. As such, one is drawn away from the consid-
eration of “faults” and frictional co-transport of ions and
water, and towards the consideration of “intentional struc-
tures” with the sole purpose of ion or water transport.
The concept of carrier-mediated transport, in which an
ion is reversibly bound to a constituent on one side of the
membrane and then released on the other side, was devel-
oped by Osterhout (1935). This appears to be an early rec-
ognition that ion transport proteins (channels or pores) have
a binding site that senses the hydrated radius and charge of
an ion, which then results in conformational changes in the
transport protein that allows permissive transport through
the membrane (Talwar and Lynch 2015). In 1968, Bertil
Hille elaborated on this concept with the recognition that
“monovalent and divalent ions including hydrogen, sodium,
potassium, calcium, and magnesium ions compete with each
other for a limited number of binding sites on erythrocyte
membranes” (Hille 1968).
Key concept seven: cells have ‘structure’
that permits swelling and shrinking
in a controlled manner and that senses
changes in cytoskeletal tension
Skeletal muscle cells have considerable structure that is
manifest in an intricate network of structural proteins that are
sensitive to mechanical deformations. The structural–func-
tional network of proteins is contiguous with contractile
proteins, surface membranes, all cellular organelles, and
other cells via networks of intra- and extracellular protein
linkages (Olsen et al. 2019). The significant development
of the concept that these structural elements are involved in
signal transduction within and between cells can be attrib-
uted to Ingber (1993), who applied the concept of tensegrity,
derived from “tensional integrity” by architect Buckminster
European Journal of Applied Physiology
Fuller in 1927, to living cell systems. The application of
tensegrity to cell systems may be controversial (Ingber et al.
2000), but the research underpinning the concept has led to
significant advances in our understanding of membrane and
cytoskeletal interactions. Cytoskeletal structure is important
in both intracellular and intercellular signal sensing, signal
transduction and communication.
As stated by Olsen et al. (2019) “The skeletal muscle
membrane is a mechanically sensitive structure that houses
multiple mechano-transducing proteins and complexes.” In
their discussion of stretch activated ion channels, they focus
on the deformation of these membrane-spanning proteins
upon muscle contraction and relaxation, and associate their
activation with intracellular anabolic responses following
the cessation of contraction (McBride et al. 2000). The roles
of stretch activated ion channels in osmotically induced cell
volume changes remain to be determined in skeletal mus-
cle cells, although their involvement in chloride channel-
mediated cell volume responses has been studied in cardiac
myocytes (Suleymanian et al. 1995; Chen et al. 1996). This
is a ‘key concept,’ as it appears important for a cell to be able
to sense its shape and volume, and perhaps also the shape
and volume of its neighboring cells.
Key concept eight: regulatory volume
decrease and increase
The initial development of the concepts of regulatory vol-
ume decrease (RVD) and regulatory volume increase (RVI)
can be attributed to Kregenow’s studies of duck erythrocytes
exposed to hypotonic (Kregenow 1971a) and hypertonic
(Kregenow 1971b) solutions. These concepts were built on
the work of predecessors, including Tosteson’s studies of
­K+ transport in human erythrocytes (Tosteson 1955) and
Dick’s studies of membrane water permeability (Dick 1959).
In describing the initial swelling and subsequent reversion
to normal volume in the presence of a hypotonic medium,
Kregenow stated: “This example of volume regulation
appears to require a membrane mechanism with receptor,
transmitter, and effector properties which produces a tem-
porary increase in the rate coefficient for the potassium leak.
In contrast, the rate coefficient for the sodium leak is either
unchanged or decreased.”
There are several important characteristics of the RVD
that were identified by Kregenow (1971a). First, the initial
rapid swelling upon incubation or perfusion with hyper-
tonic medium that is less at any point in time than would
be obtained if the cells behaved as perfect osmometers.
That fact that the initial volume change is less than would
occur by a perfect osmometer is due to the active or facili-
tated transport of osmotically active molecules across the
plasma membrane. In his RVD experiments using duck
erythrocytes, Kregenow could explain the regulatory phase
of the volume decrease by ‘passive’ ­K+ leak from the cell,
though this leak rate could be increased by inhibiting NKA
activity using ouabain. However, when cells were initially
made to shrink, the resultant RVI could not be explained
by a single membrane event, such as a sole increase in
NKA activity. Only later was the role of the sodium–potas-
sium–chloride cotransporter (NKCC) shown to be involved
in RVI responses (Hoffmann et al. 1983). The second char-
acteristic is that the reduction in cell volume is propor-
tional to the decrease in tonicity of the bathing solution.
The third characteristic is that a gradual decrease in cell
water and volume during the RVD phase can be expressed
by an exponential decay requiring between 6 and 90 min
depending on the cell type and experimental condition.
Similar but reversed responses are observed when cells
are made to shrink by incubation or perfusion with hypo-
tonic medium (Kregenow 1971b, a; Hoffmann et al. 2009).
Some of these features appear to have been first described
in skeletal muscle by a series of studies performed by
Urazaev and coworkers (Urazaev et  al. 1987; Sitdikov
et al. 1989); these studies provided the foundation for the
muscle RVI research described below.
Our present day understanding of cell
volume regulation in skeletal muscle
As may be expected the sarcolemma is heavily involved in
the regulation of cell volume and has at least three major
roles in the control system. First, mechanosensitive protein
linkages respond to both contractile and non-contractile
cell stretching or shrinking (Low et al. 1997; Olsen et al.
2019). For example, during high-intensity contraction of
other large muscle groups, the initial volume loss in non-
contracting muscle is followed by a subsequent RVI, a
regulated increase towards the initial cell volume.
Second, the sarcolemma is involved in the control of
ion, metabolite and water transport through its ion chan-
nels/transporters and aquaporins (Hoffmann et al. 2009).
This is critical as it is the concentrations of osmolytes
within the cell that ultimately determine its water con-
tent. For example, to gain volume, the cell must increase
the content and concentration of intracellular osmolytes,
thereby facilitating net influx of water, thus restoring vol-
ume and shape.
Third, regulation implies a requirement for cellular shape-
sensing, osmo-sensing, or volume-sensing (stretch or slack
tension) in conjunction with signal transduction systems that
effect appropriate ion and water transport activities. Evi-
dence for an osmosensor during induced skeletal muscle cell
volume loss was provided by experiments demonstrating the
13

involvement of TRPV2 mechanosensitive channels belong-
ing to the transient receptor potential (TRP) family (Zanou
et al. 2015). These types of signal transduction mechanisms
control channel opening and closing as appropriate (Lang
2007; Hoffmann et al. 2009; Usher-Smith et al. 2009).
Contracting muscle
The loss of water from the blood during exercise (Fig. 1)
was first noted by Ranke in canine muscle (Ranke 1865)
and thereafter by Zuntz in humans (Zuntz and Schumburg
1901). While this phenomenon was systematically studied
in 1930 (Dill et al. 1930), Dill and colleagues were unable
to make a connection between blood water loss and muscle
Fig. 4  Summary of changes in metabolic and surface membrane
events that lead to changes in intracellular osmolality of contracting
muscle cells. An increase in muscle contractile activity, particularly
during high-intensity exercise, results in rapid and large increases in
the concentrations of osmotically active metabolites within muscle
cells, resulting in the net movement of water from the arterial circula-
13
European Journal of Applied Physiology
water gain, although they referred to this relationship as
described by Ranke.
With the onset of moderate- to high-intensity con-
tractile activity, the increase in muscle cell volume is a
response to a series of concurrent and sequential metabolic
and ion flux events that result in a net increase in intracel-
lular osmolality (Fig. 4). The increased concentrations of
osmotically active molecules within contracting cells have
been measured in human and rodent muscles in different
experimental conditions for more than 100 years (Need-
ham 1971). As a result, we know that contracting muscle
cells swell, lose K­ + but gain N­ a+, ­M g2+, ­C a2+, ­C l− and
water (Fenn 1936; Fenn and Cobb 1936; Fenn et al. 1938;
Sjogaard 1983; Heigenhauser and Lindinger 1988; Kow-
alchuk et al. 1988b; Lindinger and Heigenhauser 1988),
and also increase their content of osmotically active
tion into cells and interstitium (Fig. 1). Simultaneously, the increased
rate of membrane electrical excitation results in increased intracel-
lular ion concentrations – except for ­K+ which decreases. Sarcoplas-
mic reticulum C ­ a2+ release into the cytosol also contributes to the
elevated ­[Ca2+]
European Journal of Applied Physiology
metabolites including lactate, ADP, AMP, creatine, and
Pi (Meyerhof 1947; Hargreaves and Spriet 2020). Watson
et al. (1993) showed that increased intracellular “small-
molecule osmotic pressure was the primary pressure
causing the trans-capillary water flux” that resulted in
increased muscle water content.
Contraction results from depolarization of the sarco-
lemma and T system (surface) membranes (Lindinger and
Cairns 2021) that, in turn, results in sarcoplasmic reticu-
lum release of C­ a2+, consequent activation of actin-myosin
interactions, and sarcomere shortening. Surface membrane
depolarization results in a large, rapid, and transient flux of
­Na+ into the cell. Repolarization is effected by the opening
of ­K+ channels, resulting in action potential restoration. At
the end of the action potential, the skeletal muscle cell has
gained ­Na+ and lost K­ +. With repeated contractions, experi-
mental measures indicate that the net loss of ­K+ exceeds
the net gain of ­Na+ (Kowalchuk et al. 1988b; Lindinger and
Heigenhauser 1988) despite the ongoing active extrusion
of ­Na+ by the NKA (Clausen 2003). Of interest and impor-
tance with respect to control of membrane electrical stability
(Pedersen et al. 2005), there is a net gain of ­Cl− by cells,
indicating opening of ­Cl− channels at some point during
the action potential. The net effect of the gain in ­Na+ and
­Cl− is to retain water within the cell; however, this is mark-
edly offset by the concurrent loss of cellular ­K+. This leaves
accumulation of metabolic osmolytes as the primary driver
for osmotic water flow into cells, as concluded by Watson
(Watson 1993; Watson et al. 1993).
Repeated action potentials require rapid restoration (at
least partially during high-intensity exercise) of intracellular
­Na+ and K ­ + concentrations (Lindinger and Cairns 2021).
This is orchestrated by NKA, which are abundant in and can
be incorporated into surface membranes from sub-sarcolem-
mal domains when required (Pirkmajer and Chibalin 2016).
Fig. 5  Total tissue water
(TTW), extracellular fluid
volume (ECFV), and intracel-
lular fluid volume (ICFV) in
isolated, perfused rat hindlimb
muscles at rest and after high-
intensity contractions (100 Hz)
for five minutes (Stim). High
intensity contractions resulted
in increased (p < 0.001) TTW
and ICFV in all muscles except
plantaris, and ECFV increased
(p < 0.05) in all muscle except
white gastrocnemius. Values are
means ± SD. Data recalculated
from Lindinger et al. (1987)
Sub-sarcolemmal domains proximal to NKA are rich in
glycolytic enzymes and likely creatine kinase (Dölken et al.
1975; Müller 1976; Pierce and Philipson 1985) to provide
the ATP required for sustained NKA activity. The demand
for ATP by the NKA results in very rapid hydrolysis of PCr
occurring primarily within the first 30 s of activity (Spronck
1965; Trump et al. 1996). PCr hydrolysis was the largest
single contributor to the increase in intracellular osmolality,
because its hydrolysis yields two osmotically active mol-
ecules: creatine and inorganic phosphate (Pi). Within 15 s of
intense contractile activity, the degradation of 30 mmoles/L
of PCr increased intracellular osmolality by 60 mosmol/kg
(Heigenhauser and Lindinger 1988). Within 30 s of activity,
this is augmented by the intracellular accumulation of up to
40 mmoles/L of lactate. This is despite a profound increase
in the rate of its mitochondrial oxidation (Brooks 2020) and
its net efflux across the sarcolemma via monocarboxylate
transporters (Bonen et al. 2006).
In order to provide an understanding of the net effect on
cell volume of the many simultaneous events occurring in
contracting muscle, let us examine in some detail the main
process contributing to changes in intracellular osmolality
summarized in Fig. 4. The contributions of changes in intra-
cellular osmolytes can be appreciated from data like those
obtained in blood-perfused rat hindlimb muscles (Lindinger
et al. 1987). In this study, four muscles differing in fiber
type composition were maximally stimulated using electrical
excitation of the sciatic nerve, thus causing action potentials
in all muscle cells of all fiber types (unlike voluntary mus-
cle activation). The increases in TTW, ECFV and ICFV are
shown in Fig. 5.
Measurements of the concentrations of metabolites and
ions at rest and at the end of 5 min of high-intensity contrac-
tions, and of arterio-venous differences of the major elec-
trolytes, showed that the increase in intracellular osmolality
13

resulting from increased concentrations of both PCr and
Pi is partially offset in the initial 30 s period by some K ­ +
loss (Lindinger et al. 1987). After the initial 30 s of con-
tractile activity, an increase in intracellular lactate was also
appreciable. At the end of five minutes of intense activity,
the changes in the white gastrocnemius muscle (the largest
muscle in the rat hindlimb group) showed that PCr degrada-
tion contributed 18 mosmoles/L and hydrolysis of adenylate
phosphates an additional 8 mosmoles/L and increased [lac-
tate] 22 mosmoles/L. These total ‘metabolic’ contributions
of 48 mosmoles/L were incremented by increased [­ Na+] (4
mosomoles/L) and ­[Cl−] (9 mosmoles/L) and offset by losses
of ­K+ (39 mosmoles/L) and ­Mg2+ (9 mosmoles/L). The cal-
culated net increase in intracellular osmolality in these four
hindlimb muscles at the end of 5 min was between 10 and 20
mosmole/L depending on the muscle (Lindinger et al. 1987).
As a consequence of the increase in intracellular osmolal-
ity, muscle water content and muscle cell volume increased
(Fig. 5).
With repeated bouts of high-intensity exercise,
the increase in intracellular osmolality has additional
Fig. 6  The time course of intra- and extracellular water in human
muscle during during submaximal exercise (left panel) and in
response to six minutes of maximal knee extensor contractions fol-
13
European Journal of Applied Physiology
contributions from glycogenolysis which releases bound ­K+
(Bergström et al. 1971). Additionally, the hydrolysis of ATP,
ADP, and AMP further (above that arising from PCr hydrol-
ysis) increases [Pi] while glycolysis continues at high rates
further raising [lactate], with no recovery of PCr so long as
contractile activity continues (Bergström et al. 1971; McCa-
rtney et al. 1986; Hargreaves and Spriet 2020). This results
in an increase in intracellular osmolarity in the range of 60
to 80 mosmol/kg (Kowalchuk et al. 1988b) during a time
when there has already been a significant gain in intracel-
lular water content (Lundvall et al. 1970; Kowalchuk et al.
1988b). Interestingly, the net osmotic loss of ­K+ from mus-
cle is similar to the net osmostic gain of lactate (Lindinger
et al. 1995). The contracting cells increase in volume, and
are swollen. This ‘pumped muscle,’ when measured via tape
measure, actually displays an increase in whole muscle cir-
cumference (Lundvall et al. 1970).
The best human data remain those of Sjogaard et  al.
(1985) who used 3H-labelled inulin to tag extracellular water
in vastus lateralis biopsies obtained before exercise, at 10
and 20 min of half-maximal contractions and, in a separate
lowed by 30 min of resting recovery (right panel). * indicates signifi-
cant increase from rest (time 0). Data from Sjogaard et al. (1985)
European Journal of Applied Physiology
series of experiments of maximal contractions within
approximately 30 s, three minutes and 30 min after cessation
of maximal and (Fig. 6). With submaximal exercise, there
was no consistent increase in intracellular water at 10 min,
while ECFV was increased at 10 min but not at 20 min
(Fig. 6, left panel). In this experiment, the first measure-
ment at 10 min has missed the initial rapid accumulation of
osmolytes within muscle cells (first 120 s), but the effect—
together with loss of intracellular ­K+ to the interstitium—is
detected as the increase in ECFV at 10 min. In contrast,
with maximal contractions of 6 min duration, there was a
pronounced increased in ICFV — as well as ECFV—at the
end of contraction (Fig. 6, right panel). Muscle cell volume
recovery was complete within 30 min post exercise although
ECFV remained elevated at 30 min of recovery. The initial
rapid post-exercise (at 3 min post-exercise occurring at the
9-min time point in Fig. 6, right) decrease of intracellular
water suggests a rapid decrease in intracellular osmolality
that may primarily be associated with re-synthesis of PCr
(Harris et al. 1976). While further investigation is required,
the present author hypothesizes that the recovery of intracel-
lular fluid volume is primarily metabolic, with re-synthesis
of PCr as well as oxidation and release of lactate as primary
drivers. Restoration of intracellular ion concentrations (of
mainly ­K+ and ­Mg2+) increases intracellular osmolality and
thus does not contribute to the intracellular volume loss dur-
ing the initial recovery period.
While the mechanisms responsible for the recovery of
muscle water and ions would take decades to uncover, this
phenomenon appears to have been first systematically stud-
ied by Fenn et al. (1938). We now know that upon cessation
of high-intensity contractile activity, sarcolemmal ion/water
transport and intracellular metabolic enzyme activities are
very high. Lactate continues to be lost from muscle cells
via monocarboxylate transporter 1 (MCT1) until intracel-
lular lactate concentrations are 2–4 mmol/L greater than
the extracellular concentration. Lactate also continues to be
oxidized at high rates until intracellular concentrations are
low. Very high NKA activity effects rapid cellular K ­ + entry
+
which drives extracellular and plasma K ­ concentrations to
below resting concentrations, partially restores intracellular
­[K+], and fully restores intracellular ­[Na+] to or below rest-
ing levels (Clausen 2003; Lindinger and Cairns 2021). There
is a progressive recovery of PCr, ATP, ADP and AMP, thus
restoring [creatine] and [Pi] to resting concentrations (Harris
et al. 1976; Forbes et al. 2005). During high-intensity exer-
cise interspersed with rest periods between bouts, recovery
of blood parameters requires 90 min (Lindinger et al. 1992),
and it is therefore likely that muscle also requires a similar
amount of time for osmotic and volume recovery.
Moderate- to high-intensity exercise repeated at peri-
ods through the day, and particularly over periods of
weeks’ results in multiple episodes of cellular swelling of
contracting muscle. There is strong evidence that repeated
episodes of muscle swelling signal hypertrophic and ana-
bolic processes in skeletal muscle (as reviewed by Olsen
et al. 2019).
In summary, swelling of contracting muscles performing
moderate- to high-intensity exercise is the result of increases
in intracellular and extracellular osmolytes associated with
the metabolic provision of ATP to meet the demands of the
major ATP-requiring systems, i.e. NKA, Ca-ATPase and
myosin ATPase. As sub-maximal contractions continue, the
initial volume increase is attenuated by cellular losses of ­K+
and lactate as well as partial re-synthesis of PCr. It remains
to be determined if the volume responses in contracting mus-
cle fibers are regulated, or merely passive.
Non‑contracting muscles
By way of prefacing what occurs in non-contracting muscles,
it is helpful to consider what occurs in blood during high-
intensity exercise (Fig. 1). Calculations of the total amount
of water taken up by contracting muscle cells (increase in
total intracellular fluid volume) during high-intensity exer-
cise indicate that this amount exceeds the measured decrease
in plasma volume by as much as two-fold (Lindinger et al.
1990). This result is consistent with observations in isolated,
perfused muscles (Watson et al. 1993; Ward et al. 1996).
It is also known that the extracellular fluid volume sur-
rounding contracting muscle increases (Sjogaard and Saltin
1982; Sjogaard et al. 1985). Because the calculated fluid
uptake into contracting muscle is greater than the decrease in
plasma volume, there must be a net loss of fluid from other
tissues, including non-contracting skeletal muscle (Fig. 7).
Therefore, there is a partial defense (replenishment) of
plasma volume, and hence blood volume, by the net flux of
water from non-contracting tissues into plasma (Figs. 1, 7).
This concept was demonstrated experimentally during both
a single 30 s bout of high-intensity exercise (Kowalchuk
et al. 1988a) and with four repeated 30 s bouts of exercise
(Lindinger et al. 1990). It can be concluded that during high-
intensity exercise, fluid transfer from non-contracting tis-
sues augments blood volume which will impact associated
cardiovascular responses.
During high-intensity exercise, compared to the resting
state prior to exercise, non-contracting tissues are perfused
with arterial blood with decreased water content, increased
osmolality, increased concentrations of lactate, K ­ +, ­H+,
total protein, and red blood cell content (Lindinger et al.
1992; Fig. 7). It is therefore not surprising that studies of
non-contracting muscle reported extraction of lactate and
­K+ from the arterial circulation, as well as modulation of
blood and whole body acid–base state, without substan-
tial intracellular perturbations (Kowalchuk et al. 1988a;
13

Fig. 7  Summary of cellular changes in non-contracting skeletal mus-
cle that lead to changes in intracellular volume during periods of
high-intensity exercise. The increase in arterial osmolality provides
the driving force for net water loss from non-contracting cells. A neg-
ative arterio-venous difference for plasma protein concentration seen
with high exercise intensities indicates a net loss of water from non-
contracting cells into the circulation—this would be associated with
Lindinger et al. 1990)—the intracellular distribution volume
of non-contracting cells is large and thus prevents detection
of small but physiologically relevant contributions to the
plasma volume. For example, at the end of the first two bouts
of high-intensity exercise, femoral venous plasma volume
was 127 mL less than arterial plasma volume, as calculated
from plasma protein concentrations (Lindinger et al. 1992).
Evidently, despite their state of ‘relaxation,’ non-contracting
tissues play a very important supporting role during exer-
cise. Similar compensatory plasma volume responses to
those occurring with exercise have also been observed in
response to acute blood withdrawal (20% of blood volume)
Fig. 8  The same single mouse muscle fiber (extensor digitorum lon-
gus) is shown in all panels, and the fiber remains unstimulated (elec-
trically) throughout the experiment. The red bar shows the width
of the fiber when in a normotonic solution (left panel), and the size
of the bar is unchanged in the middle and right panels. After a 90-s
exposure to increased extracellular tonicity, the cell has lost volume
13
European Journal of Applied Physiology
cell shrinking. There is also evidence (based on arterio-venous dif-
ferences) that non-contracting cells extract K ­ + and lactate from the
arterial circulation (Lindinger et  al. 1995) such that this would aug-
ment intracellular osmolality and contribute to a regulatory volume
increase (RVI). Surface membranes include the plasma membrane
and transverse tubules. NKA sodium potassium ATPase
and confirmed with subsequent plasma volume expansion
(González-Alonso et al. 2006).
It was of interest to determine if the mechanisms used
by non-contracting muscle to regulate or control its vol-
ume were similar to those employed by epithelial cells.
The prevailing thought, prior to conducting the experi-
ments, was that increasing extracellular osmolarity would
result in a passive loss of water from muscle, and that the
muscle would gradually lose volume until a new steady
state with the perfusing arterial blood was achieved. To
do this, single muscle fibers were isolated from mouse
extensor digitorum longus. After a resting period of at
least 60 min at 37 °C that allowed for cells to adhere to
(middle panel) as indicated by the cell width being reduced compared
to the red bar. After an additional 15  min of exposure to increased
extracellular tonicity, the fiber has re-gained significant volume.
The gain of volume, after the initial osmotic loss of water volume,
is termed regulatory volume increase (see Lindinger et  al. 2011 for
additional details)
European Journal of Applied Physiology
the bottom of the well, fibers were subjected to a step
increase in osmolality by injection of a small volume of
concentrated sucrose into the well (Lindinger et al. 2011).
Muscle fibers rapidly (within 15 s) lost considerable vol-
ume with a stepwise increase in extracellular osmolarity,
but subsequently regained a substantial amount of this
lost volume over time (15 min) when extracellular osmo-
larity remained elevated (Fig. 8). The initial loss of cell
volume suggests remarkably high permeability to water
(Dick 1966; Kregenow 1971b) suggesting a transient
opening of aquaporins to facilitate the water transport—
this remains to be investigated. While this RVI phenome-
non is common in many cell types, this was its first direct
demonstration in skeletal muscle (Lindinger et al. 2002)
following initial studies by Urazaev et al. (1987), Sitdikov
et al. (1989). Nonetheless, the theory that skeletal muscle
cells were capable of RVI remained controversial until
the follow-up experiments using single-cell techniques
visually showed the characteristic changes in single-cell
shape and volume (Lindinger et  al. 2011). In resting,
non-exercised muscle fibers, the RVI thus results from an
increase in cellular ion content, as has been definitively
demonstrated by Tosteson and Hoffman in erythrocytes
(Tosteson and Hoffman 1960), which causes a secondary
flow of water (Dick 1966).
Similar to epithelial cells, the most notable feature of
RVI in resting skeletal muscle is increased activity of the
NKCC when perfused with hypertonic solutions. This was
directly demonstrated in situ via perfusion with hyper-
tonic blood solutions in the absence or presence of a spe-
cific NKCC inhibitor (Lindinger et al. 2002, 2011; Gos-
manov et al. 2003b). The NKCC appears to be important
for muscle cell volume regulation even when perfused
with normotonic solutions, as inhibition of the NKCC
resulted in net water loss from muscle (Lindinger et al.
2002). This effect may reflect an altered muscle K ­ + bal-
+
ance, because the NKCC mediated ­K flux accounts for
about 15% of the NKA-mediated K ­ + flux (Lindinger et al.
2002). This action occurs in tandem with other membrane
activity. Namely, sustained NKCC activity is dependent
on the NKA, which is sensitive to the increased intracel-
lular ­Na+ concentrations resulting from increased NKCC
activity (Gosmanov and Thomason 2002; Gosmanov et al.
2002; Lindinger et al. 2002, 2011).
In summary, in resting skeletal muscle perfused with
hypertonic solutions, analogous to its environment during
high-intensity exercise, cell shrinkage results in increased
ion transport activity, bringing osmolytes into the cell to
partially restore volume. This appears to be regulated, in
that there must be a sensor for the volume loss/shrinkage;
likely the cytoskeleton. Furthermore, signal transduction
mechanisms clearly stimulate NKCC activity (Gosmanov
et al. 2003b) and, when extracellular lactate is available,
MCT activity (Lindinger et al. 2011). Both MCT 1 and
MCT 4 contribute to lactate influx into resting muscle
during high-intensity exercise; mitigating the initial
volume loss upon exposure to hypertonic solutions, and
inducing a faster and more complete RVI response follow-
ing the initial volume loss (Lindinger et al. 2011, 2013).
Future directions
In contracting muscles, it currently remains unknown if
the increase in muscle cell volume results in regulated
responses. For example, is inwardly directed aquaporin
closure and outward ion transport activation regulated to
mitigate excessive intracellular increases in water con-
tent or to restore volume, or are these merely coinciden-
tal events? Leaving aside the question of regulation, the
responses that do appear to mitigate excessive increases
and restore volume are summarized below.
It remains for future research to demonstrate if skel-
etal muscles perfused with hypotonic solutions exhibit
RVD, both in resting and contracting muscle. It is highly
likely, as with other cell types, that resting muscle can be
made to swell by perfusion with blood-like solutions of
significantly decreased (10% or more) osmolality. Blood
or blood-like is important because when inappropriate
perfusion solutions are used the results obtained cannot
readily be translated to in vivo conditions and may be of
poor physiological value. There are as yet unknown con-
stituents in plasma that are crucial to obtaining physiologi-
cal responses in cell systems including skeletal muscle
(Lindinger et al. 1986) and red blood cells (Lindinger et al.
1999). Because skeletal muscle cells are well-endowed
with the same types of ion transport and control systems
present in epithelial cells exhibiting RVD and RVI, it is
expected that resting skeletal muscle will also exhibit an
RVD response to perfusion with solutions with reduced
osmolality.
Summary and conclusion
Skeletal muscle cells can both gain and lose volume dur-
ing periods of exercise and rest. Muscles do not behave as
perfect osmometers because the volume changes are less
than predicted from the change in extracellular osmolality.
Muscles contracting at moderate- to high-intensity gain
cell volume because of increased intracellular osmolal-
ity. The main osmotically active molecules driving this
increase are creatine, inorganic phosphate and lactate with
lesser contributions from N­ a+ and C
­ l−. After a rapid initial
cell volume gain, there is a partial recovery of volume as
13

contraction continues. During moderate- to high-intensity
exercise, non-contracting muscles initially lose volume
because of increased extracellular osmolality because
blood osmolality has been increased. After the initial
cell volume loss, the cells undergo a regulatory volume
increase associated with the activation of ion transport
mechanisms. Also, after an initial rapid change in volume,
there is a gradual and partial recovery of cell volume that
is effected by ion and water transport mechanisms. The
mechanisms have been studied in non-contracting muscle,
but remain to be fully elucidated in contracting muscle.
Volume appears to be regulated in skeletal muscle because
volume, at the least, is associated with strength of contrac-
tions and with anabolic signaling.
Acknowledgements  MIL is grateful to Dr. Irena Rebalka for carefully
reviewing and editing an earlier version of this paper, and to the review-
ers who provided many valuable suggestions for improvements. MIL
also expresses many thanks to all of the former graduate studies that
have contributed to the inspiration and work for some of the research
described in the paper. Figures 1, 2, 3, 4 and 7 were created using
BioRender.com.
Author contributions  MIL conceived, wrote and edited the submitted
document in its entirety.
Funding  No funding was received to assist with the preparation of
this manuscript.
Availability of data and material  This is a review with no new data.
Code availability  Not applicable.
Declarations  9258(19)​37635-5
Conflict of interest  The authors serves on the Editorial Board for the
European Journal of Applied Physiology.
Ethics approval  Not applicable.
Consent to participate  Not applicable.
Consent for publication  Not applicable.
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