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Scand J Med Sci Sports 2005: 15: 349–380 COPYRIGHT & BLACKWELL MUNKSGAARD 2005

Printed in Denmark . All rights reserved


DOI: 10.1111/j.1600-0838.2005.00457.x

Adaptation of muscle size and myofascial force transmission:


a review and some new experimental results
P. A. Huijing1,2, R. T. Jaspers1
1
Instituut voor Fundamentele en Klinische Bewegingswetenschappen, Faculteit Bewegingswetenschappen, Vrije Universiteit,
Amsterdam, The Netherlands, 2Integrated Bioengineering for Restoration of Human Function, Faculteit Construerende Technische
Wetenschappen, Universiteit Twente, Enschede, The Netherlands
Corresponding author: Peter A. Huijing, Faculteit Bewegingswetenschappen, Vrije Universitet, Van de Boechortsstr 9, 1081
BT Amsterdam, The Netherlands. E-mail: P_A_J_B_M_Huijing@fbw.vu.nl
Accepted for publication 21 February 2005

This paper considers the literature and some new experi- number. This is very different from substantial effects
mental results important for adaptation of muscle fiber (within days) of immobilization in vivo. It is concluded
cross-sectional area and serial sarcomere number. Two that overall strain does not uniquely regulate muscle fiber
major points emerge: (1) general rules for the regulation size.
of adaptation (for in vivo immobilization, low gravity Force transmission, via pathways other than the myo-
conditions, synergist ablation, tenotomy and retinaculum tendinous junctions, may contribute to the discrepancies
trans-section experiments) cannot be derived. As a conse- reported: because of substantial serial heterogeneity of
quence, paradoxes are reported in the literature. Some sarcomere lengths within muscle fibers creating local varia-
paradoxes are resolved by considering the interaction be- tions in the mechanical stimuli for adaptation. For the
tween different levels of organization (e.g. muscle geome- single muscle fiber, mechanical signalling is quite different
trical effects), but others cannot. (2) An inventory of signal from the in vivo or in vitro condition. Removal of tensile
transduction pathways affecting rates of muscle protein and shear effects of neighboring tissues (even of antago-
synthesis and/or degradation reveals controversy concern- nistic muscle) modifies or removes mechanical stimuli for
ing the pathways and their relative contributions. adaptation.
A major explanation for the above is not only the It is concluded that the study of adaptation of muscle
inherently limited control of the experimental conditions size requires an integrative approach taking into account
in vivo, but also of in situ experiments. fundamental mechanisms of adaptation, as well as effects
Culturing of mature single Xenopus muscle fibers at high of higher levels of organization. More attention should
and low lengths (allowing longitudinal study of adaptation be paid to adaptation of connective tissues within
for periods up to 3 months) did not yield major changes in and surrounding the muscle and their effects on muscular
the fiber cross-sectional area or the serial sarcomere properties.

The need for understanding muscular adaptation pennate muscle in which the estimate has been very
often confounded by erroneously introducing angu-
Major determinants of the capability of movement lar factors into the calculation (e.g. Fukunaga et al.,
induced by a muscle activity are (1) the maximum 1996). Another important factor is the measurement
(optimal) force that can be exerted by an active of fiber or fascicle lengths at a standard muscle length
muscle and (2) the length at which optimal force is (e.g. optimum length or another standard).
exerted (muscle optimum length), as well as (3) its The muscular length range of active force exertion
length range of active force exertion. These factors is determined primarily, but not exclusively, by the
can readily be quantified in experimental research on number of sarcomeres arranged in series within the
experimental animals, but are far more difficult to muscle fibers (for a review on additional contributing
assess in humans. factors see Huijing, 2000). Unfortunately, this factor
Optimal muscle force is largely determined by the cannot readily be estimated for in vivo muscles.
physiological cross-sectional area of the muscle (Af, These prime parameters Af and serial sarcomere
usually defined at optimum length). The ratio of number are adjusted so finely to functional demands
muscle volume to (mean) muscle fiber optimum in daily life, that healthy people can use their muscles
length (or alternatively mean fiber length at muscle without much attention to the tasks to be executed.
optimum length) yields a fairly good estimate for Af. This means that as the body grows the fine-tuning to
It should be noted that this is also true for very functional tasks remains. A possible exception may

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Huijing & Jaspers
be the growth spurt during initial phases of puberty. implicit assumption is made that the contra-lateral
However, in many pathological cases, muscular muscle serving as control is not affected by the
properties may limit such easy use of muscle. For intervention. It is clear that such an assumption
example, a loss of muscle mass may occur in pathol- may be false in many cases: it is unlikely that the
ogies such as cerebral palsy (Sage, 1992; Young, use of the contralateral limb will be unchanged,
1994), spinal cord injury (Thomas et al., 1997; Castro if very substantial changes are imposed on the
et al., 1999), chronic obstructive pulmonary disease experimental limb of an animal or human. In some
(Gosker et al., 2000) as well as cardiac cachexia cases it is clear that changes in some parameters of
(Mancini et al., 1992; Gosker et al., 2000; Schulze the contra-lateral muscle may be higher than in the
et al., 2002). In such cases, muscle fibers are not experimental muscle (Heslinga & Huijing, 1993).
adapted optimally (regarding Af and/or serial sarco- (2) Comparison with muscles of other individuals
mere number) to the daily tasks, causing limited not undergoing the intervention avoids comparisons
mobility in those patients. between muscles that both may have adapted, but
The development of effective therapy requires a introduces inter-individual variance into the results.
fundamental knowledge of the mechanisms affecting Unfortunately, in this type of work, longitudinal
hypertrophy/atrophy and adaptation of the number studies studying effects and mechanisms of adapta-
of sarcomeres in series (below further referred to as tion are usually not possible.
serial sarcomere number). Also for sports sciences
such knowledge may be important to develop im-
proved training methods. Natural growth
This review will integrate, on the one hand, results
regarding adaptation of the prime parameters Af and In young animals that are growing in body size, the
serial sarcomere number from experiments per- growing bones will stretch the muscles and keep them
formed in vivo, in situ as well as in vitro, with those dynamically active at relatively higher lengths and
of mathematical modelling of muscle (bioengineer- possibly enhanced length ranges because of increased
ing) on the other hand. Such a study involves very moment arms. It is obvious that such conditions
different levels of organization, at the level of (a) the constitute a signal for adaptation of serial sarcomere
organism, (b) the limb, (c) the muscle group, (d) the number as well as Af. Generally, in healthy animals
organ (i.e. muscle) and (e) the cell (i.e. muscle fiber). for both variables sizable increases have been re-
Therefore, for the study of muscle properties an ported accompanied by increase in optimum length
integrative approach with respect to the level of (Crawford, 1954; Goldspink, 1964, 1968; Williams &
organization has recently been argued to be neces- Goldspink, 1971; Tardieu et al., 1977; de Koning et al.,
sary (Huijing, 2003), because of effects of myofascial 1987; Heslinga & Huijing, 1990; Heslinga et al., 1995).
force transmission. This type of force transmission
will also be considered below.
Immobilization of joints
Muscle kept at low length
Classical animal experimentation and some human in
vivo experiments In this experiment, sustained maximum ankle plantar
flexion in a plaster cast immobilized cat or rodent
Our, rather limited, insights into mechanisms of soleus muscle at low length was maintained (Tabary
muscular adaptation are based predominantly on et al., 1972; Goldspink et al., 1974; Williams &
these types of experiments. Several relatively simple Goldspink, 1978; Spector et al., 1982; Heslinga &
to impose experimental conditions have been used Huijing, 1993). Consistently, such experiments
to study in vivo effects on Af and serial number of yielded reductions in the number of sarcomeres in
sarcomeres, as well as functional consequences of series of m. soleus ( 25% to 40%) after several
any such adaptation. Note that in such animal weeks of immobilization. Findings consistent with
experiments the functional assessments were always this were also reported for the diaphragm for em-
made on fully dissected muscle active in situ. physematous rats (Shrager et al., 2002).
Researchers in this field are usually faced immedi- Also in m. soleus, Af decreased by 35–40% after
ately with a difficult decision: To what should the low length immobilization (Spector et al., 1982;
results of an experimental muscle be compared? Heslinga et al., 1995). As a consequence optimal force
There are usually two options, each with its own was reduced, and this force was attained near the
advantages and disadvantages. (1) Comparison with length of immobilization, i.e. at a considerable short-
the muscle within contra-lateral leg of the animal. An ening of the muscle (Williams & Goldspink, 1978).
obvious advantage is that inter-individual variance Such results have led to the classical concept that
does not affect the results. In this case the sometimes- the actual experimental condition of the muscle

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Adaptation of muscle size and myofascial force transmission
determines the processes of adaptation (Williams & An important finding of those studies on muscles
Goldspink, 1978). of the lower limb was again that the new optimum
Intact innervation does not seem to be a major length is found near the length of immobilization.
factor in this type of adaptation, as in denervated For muscles immobilized at high lengths (i.e. over
muscles immobilized at low length the serial sarco- optimum length), the increase in the serial sarcomere
mere number was reduced equally as within muscles number was not affected by muscular activity (Wil-
with intact innervation. The process of adaptation liams & Goldspink, 1978). These findings indicate
occurred only slower (Goldspink et al., 1974; Hayat that activity is not necessary for adaptation of the
et al., 1978). serial sarcomere number.
However, contrasting findings are reported also
for adaptation of serial sarcomere number, particu-
larly for highly pennate muscle. Tardieu et al. (1974) Low gravity conditions and limb suspension and bed rest
found no change in serial sarcomere number for cat Human space travel has led to a highly increased
tibialis anterior muscle after immobilization at low interest in effects of micro-gravity on skeletal muscle.
length. In contrast, Tabary et al. (1972, cat soleus On earth, this has also led to enhanced interest in the
muscle) as well as Heslinga and Huijing (1993, rat effects of limb or body suspension, presumably being
soleus) found substantially decreased serial sarco- a valid model for low gravity conditions. A plethora
meres numbers ( 20% to 40%). However, for of literature can be found indicating that skeletal
medial gastrocnemius muscle of the same rats (He- muscles are vulnerable to marked atrophy under
slinga & Huijing, 1993) immobilized at similar nor- micro-gravity (Nikawa et al., 2004). Sometimes,
malized fiber lengths they found no decrease in serial such atrophy is reported in terms of changes in
sarcomere number, but did find substantial decreases muscle volume (Akima et al., 2000) and sometimes
in optimum muscle length as well as significant as decreases in physiological cross-sectional area of
atrophy. muscle (Miyamoto et al., 1998; Wimalawansa et al.,
Immobilization at low lengths, interrupted by 1999) or muscle fibers (Roy et al., 1999a). Even short
short periods during which the muscle was kept at periods of unweighting because of micro-gravity or
high lengths, also contributed to recognizing the very limb suspension result in decreases in the cytoplasmic
high adaptive signal implicit in placing the muscle at volume-to-myonucleus ratio (Kasper & Xun, 1996).
high lengths. Gomes et al. (2004) imposed maximally Somewhat surprisingly, we have not been able to
high lengths (presumably through maximal dorsal find reports regarding effects of micro-gravity or
flexion of the ankle) only once a week for 40 min, on unweighting on variables of length–force character-
rat soleus muscle immobilized in shortened position. istics or serial sarcomere number.
This short high length exposure provided significant It is clear that, also in this area of research, effects
protection against muscle fiber atrophy. In contrast, of actual length of the muscle or tendon complex are
it was not sufficient to prevent the reduction of often disregarded. How important such effects may
muscle weight and of serial sarcomere number. Ear- be, is evident from the finding reported by Goldspink
lier Williams (1990) had shown that periods at high et al. (1986) that the much smaller atrophy of
lengths as short as 1/2 an hour daily were sufficient, extensor muscles in suspended limbs represents an
not only to prevent loss of sarcomeres, but actually underestimate of the true atrophic effect because of
to cause an increase in serial sarcomere number. high length-related protein synthesis enhancement in
those muscles. It is even conceivable that at least
some of the differential atrophy effects reported for
Muscle kept at high length
muscles of different fiber type compositions may be
The findings regarding the powerful effects of tem- related to such length effects.
porarily keeping immobilized muscle at high lengths
are consistent with the general anabolic effects of
stretching a muscle to high lengths reported by Tenotomy, myotomy or denervation of synergistic
Goldspink (1977). Therefore, it is not surprising muscles: overloading agonistic muscle
that permanent immobilization at high lengths A large body of literature may also be found on what
yielded increases in serial sarcomere number and at is called compensatory hypertrophy after tenotomy,
least the prevention of major atrophy or even in- or more often ablation, of synergist muscles. It
creased muscle fiber diameters (Crawford, 1954; should be noted that synergist tenotomy does not
Tabary et al., 1972; Tardieu et al., 1974; Williams always have the expected hypertrophy effect (Ohira,
& Goldspink, 1978). Consistent findings were also 1989). Regarding many effects, related to compensa-
reported for the diaphragm for emphysematous rats tory hypertrophy, the literature is contradictory. This
after lung volume reduction surgery (Shrager et al., is particularly true for the question of occurrence of
2002). hyperplasia. A meta-analysis of 17 studies has been

351
Huijing & Jaspers
suggested to show significant increases in muscle In any case, compensatory hypertrophy should
fiber number (hyperplasia) (Kelley, 1996). However, also be regarded with myofascial effects in mind.
previously, severe criticism had been directed (e.g.
Taylor & Wilkinson, 1986) to, at least, some of the
methods used to estimate muscle fiber number ex- Retinaculum release: changing muscle–tendon
periments. This criticism led to the conclusion that complex length and moment arm
hyperplasia has not yet been substantiated. It has been hypothesized that the serial sarcomere
On the other hand, after compensatory hypertro- number is determined by the magnitude of excursion
phy many small diameter fibers were seen in muscles performed by the muscle (e.g. Herring et al., 1984;
undergoing such hypertrophy. These fibers appear to Burkholder & Lieber, 1998; Koh & Herzog, 1998).
be new fibers arising from satellite cells. They were For a start, this seems to be quite a reasonable
not seen after irradiation, which prevents hypertro- hypothesis that may be derived from the fact that
phy by impairing activation, proliferation and/or during natural growth with increased body size, both
differentiation of satellite cells (Phelan & Gonyea, moment arms and serial sarcomere numbers increase
1997). Yet, an increased number of branched muscle substantially. Retinaculotomy is supposed to in-
fibers (Tamaki et al., 1996) may very well explain crease the moment arm (i.e. the distance between
findings interpreted as hyperplasia. In such a case, the muscle line of pull and the axis of joint rotation)
the branching of muscle fibers should be interpreted and therefore leads to enhanced excursion of the
more as incomplete fusion during a process of muscle for identical joint ranges. It also leads to
regeneration after damage rather than splitting of a changed joint angle muscle length relation: for a
muscle fibers. given joint angle the muscle–tendon complex will be
A usually very sizable increase in muscle mass and shorter. The presence of two opposing signals (short
hypertrophy of muscle fibers is a very general finding muscle, increased length range) made it an appar-
(see most references in this paragraph). An interest- ently elegant experiment to identify the most impor-
ing observation may be the following contrast be- tant parameter for adaptation of muscle size.
tween two types of results found sometimes: on the However, experiments designed to do this, yielded
one hand, we have muscle fiber type conversion to opposite conclusions: Burkholder and Lieber (1998,
slow types (i.e. an increase in percentage of type I adult mouse anterior tibial muscle) found a reduction
muscle fibers; Degens et al., 1995), as well as an of serial sarcomere number, Koh and Herzog (1998,
increasing slower myosin heavy chain (MHC) iso- young rabbits) found an increase. Such contrasting
forms and a concomitant decrease in the faster MHC findings regarding the adaptation of the serial sarco-
isoforms (Stone et al., 1996). On the other hand is the mere number indicate that either species differences
finding that isometric twitch time to peak tension may be very important or the imposed in vivo
remains unaltered. This could lead to the hypothesis conditions may not have been similar.
that the slower intrinsic velocity of contraction is
compensated by an increased number of serial sar-
Explaining some paradoxes: adaptation results are
comeres. Freeman and Luff (1982) reported such an
dependent on muscle and tendon architecture as well
increase in the number of serial sarcomeres.
as on age
The results even of ablation experiments have been
interpreted almost exclusively in simple terms of Immobilization of young growing animals
effects of overload of the remaining agonistic muscles. Immobilization of muscle of young animals at high
However, apart from myofascial effects to be dis- length yielded similar results to immobilization of
cussed below, it is already clear that the surgical adult animals at low length (Tardieu et al., 1977).
intervention itself creates many signals for adaptation: Tardieu et al. (1977) also reported how to interpret
activated fibroblasts displaying a vesicular nucleus this initially baffling result: the length of tendon of
with prominent nucleoli and an outstanding increase the young muscles immobilized at high length in-
in cytomembranes, particularly the rough endoplas- creased very much changing the experimental condi-
mic reticulum, and the Golgi complex were reported tions of the muscle belly and of the fibers from high-
for both sham-operated and experimental animals to low-length conditions during the experiment.
(Zamora & Marini, 1988). Damage, rather than an
increase in muscle activity, may play a more signifi-
Effects of hyper or atrophy in pennate muscle
cant role in at least the early activation of satellite cells
during compensatory hypertrophy (Snow, 1990). In parallel-fibered muscle or pennate muscle of a low
Such conclusions are also supported by the similar degree (i.e. muscles with the line of pulls of muscle
overall effects on muscle seen after injection of and of its fibers almost parallel) the effects of adapt-
damaging muscle compounds (Rosenblatt & Woods, ing Af and serial number of sarcomeres are indepen-
1992). dent: for example, decreasing the serial sarcomere

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Adaptation of muscle size and myofascial force transmission
number shortens the muscle and atrophy decreases optimum length. Such adaptation does not include
muscle thickness. Each one of these factors does not adaptation of serial sarcomere number.
affect the other. In contrast, because of geometrical Similar effects also explain the paradox of oppos-
effects, changes of the diameter of muscle fibers of ing results described above for retinaculum release
very pennate muscle will also affect muscle length. experiments of Koh and Herzog (1998) and Bur-
One of the first authors to consider such conse- kholder and Lieber (1998). Retinaculum release has
quences of muscle geometry in the analysis of adap- two acute effects: (1) shortening of the muscle and
tation effects was Swatland (1980). muscle fibers, because of a more direct path of the
Another paradox: even within the same young tendon from insertion to the muscle belly and (2)
adult rat, the soleus muscle adapts serial sarcomere increasing the moment arm of the muscle at the joint,
number during growth and immobilization, but the so that increased muscular length ranges are neces-
overlying gastrocnemius muscle does not. Also, this sary to move the joint through an identical angle
paradoxical finding was resolved by taking into range as prior to the retinacular release.
account the geometric effects of very pennate gastro- In their argumentation, Koh and Herzog failed to
cnemius muscle (Heslinga & Huijing, 1993; Heslinga take into account additional effects of muscle atro-
et al., 1995). This means that during the period of phy, which they reported for the experimental leg
immobilization, atrophy released the muscle from the with respect to the control muscle. Following the
low fiber length-related signal to remove sarcomeres argumentation presented above, we draw the follow-
in series. This happens as follows: the muscle is kept ing conclusion: because of major atrophy, the muscle
in the maximally in vivo shortened position with fibers of rather pennate tibialis anticus (TA) muscle
short fiber and sarcomere lengths. In an unrestrained were active at higher lengths than before the atrophy.
muscle, progressive atrophy would lead to further Therefore, one should expect adaptation, i.e. an
muscle belly shortening without changing fiber and increased serial sarcomere number, based on effects
sarcomere lengths, exclusively because of the geo- of high fiber lengths, instead of causally relating
metric effect. If the muscle length is the restrained increased serial sarcomere number and the enlarged
variable, the muscle belly cannot shorten because of muscular excursion. Results for the adult mouse
atrophy, but can exclusively accommodate the geo- muscle with resected retinaculae (Burkholder & Lie-
metric effect by lengthening its fibers and sarcomeres ber, 1998) confirm such reasoning: in this case the
(Fig. 1). If this effect of atrophy is large enough to physiological cross-sectional area (Af) was not chan-
bring the fibers near their optimum length, we should ged, and therefore any confounding change in the
expect the net long-term effects of immobilization at relation of muscle length and muscle fiber length is
not likely.
Therefore, the finding that the enlarged excursion
of these muscles was accompanied by a reduction in
the serial sarcomere number seems to support the
notion that excursion per se is not a most important
regulator of the serial sarcomere number. In agree-
ment with previous literature on muscular adapta-
tion, the specific conditions of the experiment of
Burkholder and Lieber, being the lower length of
muscle fibers, because of retinacular release must
have been the determining factor for adaptation of
serial sarcomere number.
A major lesson to be drawn from these paradoxes
is that the conditions of the muscle fibers and not of
the muscle–tendon complex or muscle seem to de-
termine the adaptation effect. This means that always
effects of serial elasticity, effects of pennation, etc.
and changes thereof have to be taken into account
Fig. 1. Schematic representation of effects of atrophy and
hypertrophy and immobilization on lengths of muscles and before adaptation mechanisms can be adequately
fibers of pennate muscle. (a) Unrestrained condition. Note studied.
that pennate muscle will drastically change its length with
changes in fiber diameter because of atrophy or (hyper-)
trophy. (b) Immobilized condition. Atrophy would lead to a
shortened length in pennate muscle. Because of the restraint Molecular mechanisms of adaptation of muscle size
of immobilization the muscle can only keep its length by
lengthening muscle fibers. The arrow indicates immobilized Despite the controversies shown by the classical
length. experiments, such experiments have taught us that

353
Huijing & Jaspers
at least some physiological conditions are able to overload by removal of synergistic muscles (Allen
trigger adaptation of muscle size. However, this is et al., 1995; Roy et al., 1999b). The satellite cells, being
only a limited aspect of the unravelling of the muscle stem cells located between the basal lamina
mechanisms underlying the regulation of muscle and the sarcolemma (Mauro, 1961), are sources
size. Advances in molecular biology have facilitated for additional myonuclei (Moss & Leblond, 1971).
the characterization of relevant signalling at the Nevertheless, induction of compensatory hypertro-
cellular level (i.e. muscle fiber), as well as character- phy and hypertrophy in response to weight bearing
ization of biochemical pathways downstream to the at high length, is possible also without proliferation/
signals applied to the muscle fibers. fusion of satellite cells (Dunn et al., 1999; Lowe &
Adaptation of muscle fibers is always the net effect Alway, 1999; Rommel et al., 2001). In partial agree-
of the dynamics of synthesis and degradation of ment with this, Kadi et al. (2004) reported increased
proteins that constitutes the force generating and fiber cross-sectional area in humans because of heavy
passive elements of the muscle fiber. However, it resistance training, without an increase in the num-
should be kept in mind that in muscular adaptation ber of myonuclei, despite the fact that the number of
not only the muscle fiber needs to adapt to new satellite cells had increased.
circumstances, but, in a coordinated fashion, also the In vivo, rat and quail muscle hypertrophy in
basal lamina as well as collagen fiber reinforced response to an increase workload or continuous
extracellular matrix (ECM) components (the endo-, weight bearing (at high length) is accompanied by
peri- and epimysial stroma of the muscle, as well as proliferation of satellite cells and fusion with the
its aponeuroses). hosting muscle fiber (Winchester et al., 1991; Carson
Nevertheless, we will presently focus on muscle & Alway, 1996; Phelan & Gonyea, 1997). The
fiber processes. importance of satellite nuclei incorporation during
hypertrophy is further indicated by the lack of
compensatory hypertrophy of muscles g-radiated
Protein synthesis prior to the hypertrophic stimulus (Rosenblatt
In a theoretical analysis of changes in the rate of et al., 1994; Phelan & Gonyea, 1997; Barton-Davis
muscular protein synthesis, the elements contributing et al., 1999). On the basis of the literature cited we
to such changes may be distinguished according to conclude that it is likely that activation of satellite
two locations: outside the muscle fiber and inside the cells is very important for the induction of hyper-
muscle fiber. The former involves changes of satellite trophy. However, conditions, for which this may not
cell activity and nucleus donation to the muscle fiber, be the case, deserve further research.
as well as changing sarcolemmal receptor sensitivity Particularly for postnatal growth and hypertrophy
for growth factors. The latter involves changing the of mature muscle, several growth factors have been
quantity of mRNA and a change in the rate of identified in muscle, which are likely to be involved in
translation of mRNA. stimulating proliferation and fusion of satellite cells:
(1) during postnatal growth, the hepatocyte growth
Extracellular events factor (HGF) is expressed in the rat extensor digi-
torum muscle, but not in the mature muscle (Jen-
The number of nuclei within a muscle fiber may nische et al., 1993), (2) weight bearing at high length
increase after proliferation of satellite cells and sub- enhances expression of basic fibroblast growth factor
sequent fusion with the hosting muscle fiber. Note (bFGF) in the wing muscle of the chicken (Mitchell
that muscle fibers may also lose myonuclei by DNA et al., 1999). (3) Immobilization of rabbit muscle at
degradation. For a given rate of transcription of high length (Yang et al., 1997) or that in combination
mRNA per myonucleus, a change in the number of with increased muscle activation (McKoy et al.,
myonuclei within the sarcoplasm implicates a change 1999) stimulates expression and secretion by muscle
in total capacity and rate of mRNA transcription. fibers of insulin-like growth factors (IGFs): IGF-1
and the IGF-1 splice variant called mechano-growth
Activation of satellite cells, satellite cell proliferation factor (MGF). The capability of such growth factors
and fusion with the hosting muscle fiber. Within to bind to receptors on the satellite cell plasmalemma
mammalian as well as amphibian muscle, the number and stimulate proliferation and differentiation of
of myonuclei per unit fiber length is proportional to muscle precursor cells has been shown for in vitro
the cross-sectional area of the muscle fibers (Roy et cultures of myoblasts and satellite cells (for compre-
al., 1999b; Jaspers, 2002). This indicates that within hensive reviews see Florini et al., 1996; Grounds,
healthy muscle fibers in vivo the volume of cytoplasm 1998; Hawke & Garry, 2001; Adams, 2002; Spangen-
per myonucleus is strictly regulated. Such regulation burg et al., 2002). Whether these growth factors
was also shown after experimental hypertrophy, of act independently, or in synergy, remains a matter
rat as well as cat muscle, in response to functional of debate.

354
Adaptation of muscle size and myofascial force transmission
The results of some of the in vitro experiments other (Roghani et al., 1994; Lin et al., 1999). Other
indicate the capability of IGF-1, bFGF or HGF to ligands may cause such effects as well: the effects of
stimulate cell proliferation directly (Hawke & Garry, IGF-1 are modulated by one of six identified IGF-1
2001). In contrast, there are also reports that IGF-1 binding proteins, which affect the affinities of the
and HGF are unable to induce myoblast prolifera- IGF-1 receptor (IGFR1) and IGF-1 for each other
tion themselves, but require unidentified serum com- (for a review see Jones & Clemmons, 1995; Florini et
ponents to allow myoblast division to progress al., 1996). IGF-1 binding proteins do associate with
(Florini et al., 1996; Grounds, 1998). These data proteins at the cell surface or further away within the
are consistent with the finding that in vitro the ECM and by this increase the local concentrations of
simultaneous presence of bFGF and IGF-1 in a IGF-1 in the vicinity of the IGFR1 (Jones & Clem-
serum-free medium yielded remarkably higher rates mons, 1995).
of satellite cell proliferation, than when these cells
were exposed to these factors independently (Doumit Intracellular events
et al., 1993; Allen et al., 1995). Such findings indicate
For a given number of myonuclei per muscle fiber,
that proliferation of satellite cells is regulated by
the rate of transcription is affected by the presence or
complex interactions between different growth fac-
absence of transcription factors, which either facil-
tors. The feature, that may explain why the mechan-
itate or inhibit transcription of muscle-specific genes.
isms underlying these interactions have remained
A higher quantity of mRNA implicates the presence
undiscovered as yet, is that in order to be successful
of more templates for mRNA translation and hence
most cultures require a medium, which is supplemen-
higher capacity and rate of synthesis of the corre-
ted with serum of unknown composition regarding
sponding protein.
growth factors and other peptides.
During hypertrophy of rat plantar flexor muscles
It should be noted that Nitric oxide synthase
in response to either increased activation or removal
(NOS) producing nitric oxide (NO) is also involved
of synergistic muscles, MHC mRNA was increased
in stimulating proliferation and fusion of satellite
(Periasamy et al., 1989; Wong & Booth, 1990). In
cells (Anderson, 2000; Anderson & Pilipowicz, 2002).
addition, induction of hypertrophy of chicken ante-
Myostatin, opposing effects by the factors promot-
rior latissimus dorsi muscle by continuous weight
ing proliferation and fusion with the hosting muscle
bearing (at high length) was accompanied by an
fiber, has been identified as an inhibitor of satellite
increase in a-skeletal actin mRNA (Carson & Alway,
cell proliferation (Thomas et al., 2000; McCroskery
1996; Carson, 1997).
et al., 2003). This correlates with the finding that hind
The rate of translation of mRNA into peptides is
limb suspension is accompanied by enhanced myos-
determined by the number of ribosomes per mRNA
tatin expression and a substantial reduction of the
as well as the rate of translation per unit mRNA.
number of myonuclei (Carlson et al., 1999; Kawada
et al., 2001). Hypothetically, myostatin prevents
Biochemical-signalling pathways involved
replacement of degrading myonuclei, causing a re-
duction of the capacity and rate of the transcriptional The biochemical-signalling pathways (Fig. 2), which
machinery. Accordingly, myostatin may be viewed as are likely to be involved in muscular adaptation, will
a mediator of muscle atrophy induction. However, it be discussed briefly below.
should be noted that such a hypothesis was not
confirmed for myostatin-deficient mice, since they Regulation of transcription of mRNA. In general,
lost more muscle mass during hind limb suspension two groups of pathways are distinguished: the cal-
than wild types (McMahon et al., 2003). Therefore, cium/calmodulin-dependent pathways and the mito-
the results regarding a role of myostatin in the gen-activated protein kinase (MAPK) pathways.
induction of atrophy remains ambiguous. Both are involved in the transcriptional regulation
Therefore, it must be concluded that the absence of of the adaptation of muscle size.
factors promoting satellite cell proliferation is likely
to be involved in atrophy induction. Calcium/calmodulin signalling. Intracellular
[Ca21] regulates the activity of calcineurin, a serine/
Regulation of receptor binding of growth factors. threonine phosphatase and calcium/calmodulin pro-
Heparan sulfate is one of the macromolecules of the tein kinase (CaMK) (Schulman, 1993; Klee et al.,
ECM that structures water around itself and causes a 1998). During both immobilization at high muscle
less than full fluid consistency of the ECM. The length and enhanced muscle activity, the sarcoplas-
sulfated glycosaminoglycan branches of heparan mic [Ca21] are expected to be elevated. In the case of
sulfate bind growth factors. An effect of binding of high muscle fiber length, Ca21 may enter the muscle
bFGF to heparan sulfate is to increase the affinity of fiber via stretch-activated channel within the sarco-
the plasmalemma FGF receptor and bFGF for each lemma, whereas increased activity implicates higher

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Huijing & Jaspers

Fig. 2. Schematic representation of major biochemical-signalling pathways involved in the regulation of muscle size. Two
neighboring muscle fibers are drawn schematically with their sarcolemma and basal lamina (BL, thick solid lines). The type I
collagen fiber reinforcement (e.g. endomysium) of the extracellular matrix (ECM) is not shown for reasons of clarity (for this
structure and details of BL, see Fig. 3). The abbreviations written at the location of the ECM indicate growth factors or
cytokines that have been secreted there by the muscle fibers. Growth factors are proteins with hormone-like functions.
Cytokines are glycoproteins (i.e. compounds consisting of mostly proteins, but with some carbohydrates as well) with such
functions. Major growth factors and cytokines are: insulin-like growth factors (IGF), basic fibroblast growth factor (bFGF),
hepatocyte growth factor (HGF), mechano-growth factor (MGF)and myostatin, as interleukin-4 (IL-4). They bind to a
number of sarcolemmal receptors and affect chemical-signalling pathways by doing so. Overviews of key pathways that are
activated by effects of mechanical strain and/or increased contractile activity are shown for major effects. Note that these effects
are initiated at the basal lamina and sarcolemma, the latter indicated by the chain-like elements: (I) effects on the quantity of
transcriptional machinery. By fusion of satellite cells (SC) with the muscle fiber the number of nuclei is increased (cf.
‘‘Molecular mechanisms of adaptation of muscle size’’), which affects the overall rate of DNA transcription, even if this rate
per nucleus is constant. (II) Given a set number of nuclei, all pathways leading to altered rates of DNA to mRNA transcription
are indicated in block II. The affected transcription rates to be considered are those of mRNA related to the synthesis of
muscular proteins directly (either contractile or cytoskeletal) or indirectly in two ways (a) via mRNA production for growth
factors or cytokines or (b) transcription factors. This block contains the major pathways described individually in ‘‘Molecular
mechanisms of adaptation of muscle size’’. Note that PKC pathways are activated by the release of calcium from the
sarcoplasmatic reticulum directly or via phospholipids in the sarcolemma. For explanation of the abbreviations refer to the text
in these sections. (III) The pathways affecting the translation of mRNA into proteins are grouped in block III. This block
contains the pathways described individually in ‘‘Molecular mechanisms of adaptation of muscle size’’. For explanation of
these abbreviations refer to the text in these sections. SR indicates the sarcoplasmatic reticulum releasing Ca ions into the
cytoplasm. rRNA represents ribosomal RNA, which constitute the body of ribosomes, which are the locus of mRNA to
protein translation.

or more frequent release of Ca21 from the sarcoplas- period of weight bearing at high length (Fluck et al.,
mic reticulum. 2000).
Inhibition of calcineurin-activity-induced atrophy Downstream targets of the calcineurin and CaMK-
in mouse plantaris muscle, during compensatory signalling pathways are transcription factors, such as
hypertrophy as well as during recovery from hind the nuclear factor of activated T cells (Dolmetsch
limb suspension blocks the hypertrophic response et al., 1997; Bassel-Duby & Olson, 2003), myocyte
(Dunn et al., 1999; Mitchell et al., 2002). This enhancer factor 2 (MEF2; Passier et al., 2000; Wu et
suggests that calcineurin is required for the induction al., 2001) and the GATA transcription factors (Mu-
of hypertrophy. The requirement of enhanced activ- saro et al., 1999). Note that GATA represents a
ity of CaMK in the adaptation of muscle size has not particular sequence of nucleotides within the DNA.
been indicated as yet, but is suggested as the activity Specific GATA transcription factors bind to this
of CaMK was increased in chicken muscle after a sequence to promote gene transcription.

356
Adaptation of muscle size and myofascial force transmission
Activation of calcineurin and CaMK causes confor- Activated MAPK will phosphorylate and activate
mational changes to these transcription factors, which transcription factors such as c-fos, c-jun, c-myc and
either activate them or stimulates their translocation Elk1, as well as other kinases, which affect SRF
from the cytoplasm to the myonucleus. Both effects activity and a-skeletal actin expression (Davis,
cause enhanced gene transcription, because activation 1993; Ruwhof & van der Laarse, 2000). Generally,
and translocation of transcription factors facilitate activation of the MAPK pathways during hypertro-
their binding to promotor regions of muscle genes phy is mediated via (1) receptor binding of growth
and by doing so enhance the rate of transcription. factors (e.g. IGF-1) (Florini et al., 1996), (2) activa-
Transcription of MHC and a-skeletal actin is tion of protein kinase C (PKC) or (3) integrin
reported to be affected by such transcription factors (Carson & Wei, 2000) or dystroglycan signalling
(e.g. Maeda et al., 2002; Sepulveda et al., 2002; Giger (Rando, 2001).
et al., 2004; McCullagh et al., 2004). This could be Binding of insulin or IGF-1 to their receptors
either a direct effect, or alternatively an indirect results in phosphorylation of the insulin receptor
effect. The indirect effects occur through stimulation substrate (IRS-1) and the Src homology containing
of the expression of other types of transcription protein (ShC), both which serve as multi-component
factors, such as the serum response factor (SRF) docking platforms for proteins that contain SH2
and the myogenic regulatory factors Myf5, MyoD, domains. Once bound to IRS-1 or ShC, these pro-
myogenin and MRF4 (Carson & Alway, 1996; Bas- teins are able to activate the GTPase called Ras
sel-Duby & Olson, 2003; Davis et al., 2003). This last (McCormick, 1993), which in turn activates all three
group of transcription factors is involved in regula- different MAPK pathways (Davis, 1993; Florini et
tion of promotor activity of muscle-specific genes al., 1996). In vivo, inhibition of the MAPK/ERK
(e.g. Carson et al., 1995). pathway within rat plantaris muscle prevents IGF-1-
In addition, some of these transcription factors induced hypertrophy of muscle (Haddad & Adams,
also enhance expression of growth factors such as 2004), suggesting a crucial role for this pathway in
IGF-1 (McCall et al., 2003) and cytokines (i.e. the regulation of muscle size.
glycoproteins with hormone-like functions) such as Rat soleus muscle can be genetically modified
interleukin-4 (Horsley et al., 2001). These growth causing permanent expression of activated Ras
factors are not only involved in stimulating prolif- (Murgia et al., 2000). The importance of MAPK
eration of satellite cells, but may also affect other activity in the regulation of muscle fiber size is further
signalling pathways. For example, binding of IGF-1, indicated by results of experiments in which such
to the IGFR1, stimulates transcription and transla- modified rat soleus muscle, that was also denervated,
tion of mRNA via the MAPK (see ‘‘MAPK signal- was prevented from atrophy (Murgia et al., 2000).
ling’’) and the phosphatidylinositol 3 kinase (PI3K)– Other mediators of the MAPK activity are
mammalian target of rapamycin (mTOR) pathway grouped within the PKC family (see for reviews,
(see ‘‘Control mRNA translation’’). Jalili et al., 1999; Ruwhof & Van der Laarse, 2000)
Whether the Ca21/calmodulin-activated pathways that has been shown to be involved in the induction
play a crucial role in the induction of muscle hyper- of cardiac hypertrophy (Jalili et al., 1999). Although
trophy is subjecta of controversy (e.g. Dunn et al., compensatory hypertrophy of skeletal muscle is
1999; Bodine et al., 2001b; Mitchell et al., 2002; accompanied by increased PKC activity (Richter &
Bassel-Duby & Olson, 2003). Inhibition of the calci- Nielsen, 1991), its role in the regulation of skeletal
neurin activity in skeletal muscle during compensatory muscle fiber size is not established as yet.
hypertrophy or recovery from disuse-induced atrophy Integrins and dystroglycans are trans-sarcolemmal
has been shown to block the hypertrophic response proteins that connect the cytoskeleton and ECM of
(Dunn et al., 1999; Mitchell et al., 2002). However, muscle fibers (see Fig. 3). Dystroglycans are known
this finding could not be confirmed (Bodine et al., to be associated with the focal adhesion kinase
2001b). To answer the question if these differences in (FAK) (Rando, 2001). However, there is clear evi-
experimental results are because of differing experi- dence that binding of the extracellular domain of an
mental conditions, further investigation is required. integrin to ligands, such as laminin or fibronectin,
stimulates the formation of the so-called focal adhe-
MAPK signalling. Three main branches are sion complexes (FACs) at the cytoplasmic end of the
generally distinguished for MAPK signalling: integrin. Mechanically loading integrins stimulates
activation of FAK and the small GTPase family
Rho, which in turn initiate the cascade of activation
(B1) the c-Jun N-terminal protein kinase, of the MAPK pathways via the GTPase Ras located
(B2) the extracellular regulated kinase (ERK) and at the FAC (Carson & Wei, 2000; Ruwhof & Van der
(B3) the 38 kDa stress-activated protein kinase (p38) Laarse, 2000). MAPK activity, in rat calf muscles
pathways. in situ, is rapidly increased in response to mechanical

357
Huijing & Jaspers
Compensatory hypertrophy of rat plantaris mus-
cle, after removal of a synergistic gastrocnemius
muscle, is accompanied by enhanced activity of
Rho (McClung et al., 2003). In contrast, overload
of chicken anterior latissimus dorsi muscle induced
by weight bearing at high muscle length was shown
to increase the FAK activity (Fluck et al., 1999).
Note that unloading of the rat plantar flexor muscles
by hind limb suspension reduced FAK activity
(Gordon et al., 2001) as well as the Rho concentra-
tions (McClung et al., 2004). Although direct evi-
dence for integrin-mediated MAPK signalling in
the induction of hypertrophy or adding of serial
sarcomeres is lacking, these findings implicate such
involvement.
Recently, indications of local differences in gene
expression according to location of muscle fibers
within the muscle have been shown in rat medial
gastrocnemius muscle in response to passive and
active loading. Constant mechanical loading of the
passive dissected in situ muscle (3 N/g muscle mass),
as well as sinusoidal loading yielded fairly acutely
(i.e. within 5 min) a substantially higher MAPK
activity within the proximal muscle fibers than within
distal muscle fibers (Csukly et al., 2002). Such local
differences could not be related to local difference in
muscle fiber type because other muscles (as soleus,
extensor digitorum longus (EDL) and plantaris mus-
cles) showed sensitivity of MAPK similar to the
Fig. 3. Schematic view of supramolecular organization of overall effect for medial gastrocnemius muscle. In-
connections between the cytoskeleton and the endomysium. stead, Csukly et al. ascribed the differences to effects
There are two parallel systems of connections, which have
the following elements in common: the cytoskeleton (1) that
on compartment tension because of local differences
surrounds the sarcomeres within myofibrils and is connected in muscle geometry. It should be noted, however,
by desmin filaments (2) to sub-sarcolemmal actin filaments that substantial differences in mean sarcomeren
(3). Such connections occur at least at the level of the Z- length between proximal and distal muscle fibers
disks, but may also exist at the M line. These actin filaments are reported (Zuurbier & Huijing, 1993; Jaspers
(which are different from the thin filaments of the sarco-
meres) are connected via two types of system-specific mole-
et al., 1999). These mean fiber sarcomere differences
cules to two system-specific types of trans-sarcolemmal are opposite to the presumed distribution of tension
molecules. These trans-sarcolemmal molecules are con- over intramuscular compartments and therefore not
nected to the laminin of the basal lamina (8, which in muscle likely to be the simple explanation of the differences
is also called merosin). Laminin is connected to collagen type in MAPK activity.
IV (a non-fiber forming collagen type) of the basal lamina.
The connection between the basal lamina and the collagen
In any case, a crucial role for the MAPKs is
fibers of the endomysium (10) is made by glycoproteins. indicated in the induction of hypertrophy. However,
System-specific elements are: (a) the dystrophin–sarcoglycan to answer the question if this occurs directly by stimu-
system. For this system the connection between sub-sarco- lating the transcription of muscle-specific mRNA’s
lemmal actin and laminin is made by dystrophin (4), which is or indirectly by enhancement of the expression of
connected to the trans-sarcolemmal sarcoglycans (6). (b) The
talin–integrin system (also called the focal adhesion com-
growth factors requires further investigation is re-
plex). Talin (5) connects the sub-sarcolemmal actin filaments quired.
via the integrins (7) to laminin. These systems are not fully
independent, because, since dystrophin and talin also bind Control mRNA translation. The regulation of trans-
with high affinity (Senter et al., 1993), dystrophin may form lation of mRNA involves three phases:
a link with the integrin and dystroglycan-based systems.
 initiation of translation,
 elongation of the peptide chain and
stimuli (within o10 min) (Martineau & Gardiner,  termination of translation.
2002). This effect is quantitatively related to passive
and active tensions exerted by these muscles (Marti- Changing the translational capacity (i.e. number of
neau & Gardiner, 2001, 2002). ribosomes) and/or translational rate regulates adap-

358
Adaptation of muscle size and myofascial force transmission
tation of muscle size. The latter of these two is yields enhanced activity of eIF2B through inhibition
particularly determined by the initiation of transla- of GSK3b.
tion of the available mRNA and the speed of Furthermore, the activation of the PI3K–mTOR
elongation of the peptide chain (Nader et al., 2002; pathway may also stimulate peptide-chain elongation
Bolster et al., 2003). as activation of this pathway results in decreased
During muscle hypertrophy in response to immo- phosphorylation of eukaryotic elongation factor 2,
bilization at high length in combination with elec- which increases the speed of peptide-chain elonga-
trical stimulation, the total RNA content increases tion. As hypertrophy in response to immobilization
rapidly (Goldspink, 1977; Goldspink et al., 1995). at high lengths and increased activity is accompanied
After tenotomy of rat gastrocnemius muscle, actino- by enhanced expression of IGF-1 (McKoy et al.,
mycin-D blocking of RNA synthesis was reported to 1999; Goldspink, 2003; Hameed et al., 2003) this
prevent compensatory hypertrophy of plantaris and pathway is likely to be an important determinant of
soleus muscle (Goldberg & Goodman, 1969). As the the induction of protein synthesis.
ribosomal RNA (rRNA), which forms the basis for Disuse-induced atrophy may also be regulated
attachment and subsequent translation of the mRNA by lowering the capacity or rate of translation of
template, constitutes more than 80% of the total mRNA. Total and phosphorylated Akt are decreased
RNA, blocking the increase in total RNA would after denervation-induced atrophy. Injection of a
predominantly block an increase in rRNA. Accord- plasmide (i.e. circular DNA) designed to perma-
ingly, Nader et al. (2002) argued that the lack of nently express an active form of AKT in denervated
hypertrophy by the blocking of RNA synthesis mouse tibialis anterior (i.e. bypassing the control of
was because of a lack of rRNA synthesis capacity. Akt) showed a substantial reduction in atrophy after
An increase in ribosomal number is thought to be denervation (Bodine et al., 2001b). In addition,
essential for the induction of hypertrophy. How- muscle unloading is associated with a decrease in
ever, this rationale should be viewed with some both mTOR (Reynolds et al., 2002) and P70S6
skepticism as this argumentation only holds true kinase phosphorylation (Bodine et al., 2001b), which
when the mRNA content is not limiting the rate of implicate a decreased rate of translation. Other
translation. downstream regulators of translation are the inhibi-
In addition, several cofactors of the ribosomal tory factors of translation 4E-BP-1 and EF2 kinase.
proteins, such as the eukaryotic initiation factors, The mRNA expression of these factors is elevated
elongation factors and binding proteins, have been during different atrophy models (Jagoe et al., 2002;
identified. These cofactors are involved in the regula- Stevenson et al., 2003).
tion of initiation of the peptide chain as well as of the
speed of elongation of the peptide chain. Regarding NO. NO is a free radical produced ubiquitously
adaptation of muscle size, activity of these cofactors by NOS. Three different isoforms of NOS have been
is affected by IGF-1 or insulin via the PI3K–mTOR identified (Reid, 1998; Stamler & Meissner, 2001):
pathway. Therefore, they are likely to be major type I isoform (also called neuronal NOS or nNOS),
determinants of the rate of protein synthesis (Bodine type II (inducible or iNOS) and type III NOS
et al., 2001b; Rommel et al., 2001; Pallafacchina et al., (endothelial NOS or eNOS). Both nNOS and
2002). eNOS are permanently expressed in skeletal muscle
Details about such signalling pathways have been and their activity is mediated by interaction with
reviewed extensively (Shah et al., 2000; Nader et al., calcium and calmodulin (Nathan & Xie, 1994).
2002; Bolster et al., 2003). Briefly, binding of IGF-1 Within skeletal muscle, nNOS is located below the
or insulin to the IGFR1 at the outside of the sarcolemma and associated with the dystroglycan
sarcolemma recruits IRS-1, which in turn activates complex (Brenman et al., 1995; Gossrau, 1998). In
PI3K–mTOR pathway. Downstream target of PI3K contrast, iNOS is located in the cytoplasm and eNOS
is the protein kinase B (called PKB or Akt), which on is particularly present within the mitochondria (Bates
phosphorylation activates mTOR and inhibits glyco- et al., 1996).
gen synthase kinase 3b (GSK3b). Activated mTOR Several experiments indicate a role of NO in the
does phosphorylate p70S6 kinase (p70S6K) as well as regulation of adaptation of muscle size. Increased
the binding protein for the eukaryotic initiation muscle activity is accompanied by increased concen-
factor 4E (4E-BP1). When phosphorylated, both trations of nNOS as is shown during electrical
p70S6K and 4E-BP1 promote the translation of stimulation of the rabbit EDL and TA muscle
mRNA. Apart from its function in the glycogen (chronic stimulation: 3 weeks) (Reiser et al., 1997)
synthesis enzyme cascades, activated GSK3b phos- as well as rat soleus muscle (10 min) (Balon &
phorylates the eukaryotic initiation factor 2 binding Nadler, 1997). In addition, reloading of rat soleus
protein (eIF2B) resulting in inhibition of initiation of muscle after a period of hind limb unloading was
translation. Activation of Akt, therefore, indirectly accompanied by an increase in nNOS (Tidball et al.,

359
Huijing & Jaspers
1998). Furthermore, release of NO from rat soleus The calpain system
muscle, kept in vitro at high length for 2 min in the
Calpains are calcium-activated cysteine proteases,
presence of calcium, was significantly higher than
which in muscle tend to be concentrated at the Z-
that from muscle maintained at passive slack length
disk of the sarcomeres (rat soleus muscle, Kuma-
at similar calcium levels (Tidball et al., 1998), sug-
moto et al., 1992). Calpains are particularly involved
gesting that the activity of NOS is also affected by
in the degradation of cytoskeletal proteins (Huang &
mechanical stimuli. Indications of the requirement of
Forsberg, 1998) and by doing so may make the
NO in the induction of hypertrophy and addition to
myofibrillar proteins accessible for the proteasomes.
the serial sarcomere number have been found (e.g.
A period of either unloading or denervation of rat
Koh & Tidball, 1999; Smith et al., 2002). Inhibition
soleus muscle increases the expression of calpains
of nNOS activity during compensatory hypertrophy
within the muscle (Taillandier et al., 1996; Haddad et
of rat plantaris muscle attenuates the induced hyper-
al., 2003). Inhibition of one of the calpains leads to
trophy (Smith et al., 2002). During remobilization
substantial attenuation of the atrophy (Tidball &
after a period of immobilization of the muscle at low
Spencer, 2002) illustrating their relevance in the
length, blocking of nNOS activity has been shown to
induction of muscular atrophy.
inhibit the addition of sarcomeres in series in rat
soleus muscle (Koh & Tidball, 1999).
Taken together, these findings do suggest that NOS The lysosomal proteases
is relevant in the induction of hypertrophy and The organelles called lysosomes are single membrane
addition of sarcomeres in series. The intracellular globular systems that contain hydrolytic enzymes.
biochemical mechanisms via which NOS affects pro- Lysosomes carry hydrolases that degrade nucleo-
tein synthesis and degradation are not well under- tides, proteins (e.g. cathepsins and collagenases),
stood. NO may be involved in the prevention of lipids, phospholipids polymers into their monomers,
protein degradation. and also remove carbohydrate, sulfate, or phosphate
groups from molecules. These hydrolases are parti-
Protein degradation cularly active in an acid environment, which is
fortunate because, if they leak into the cytoplasm
Degradation of proteins is contrasted to their synth- at physiological pH (  7.2–7.4), they are not likely
esis in the following way: to do much damage. Ubiquitination of sarcolemmal
Synthesis involves genetic expression of the muscle proteins such as receptors and channels make them
proteins themselves (transcription and mRNA trans- targets for the lysosomal systems, which will reduce
lation), as well as expression of cofactors and activa- their number and thus their involvement in the
tion of those cofactors, leading to enhanced or induction of protein synthesis (Taillandier et al.,
decreased expression of the relevant muscle proteins. 1996; Jackman & Kandarian, 2004).
Therefore, a decrease in synthesis rate of the Increased activities of cathepsin have been shown
relevant proteins at constant rate of catabolism in soleus and extensor digitorum muscle from hind
would lead to a diminishing size of muscle. limb suspended rats (Goldspink et al., 1986) suggest-
In addition, degradation is regulated by the activ- ing their possible involvement in the induction in
ity of proteolytic enzymatic pathways and by expres- atrophy.
sion of cofactors and activation of these cofactors
leading to enhanced or decreased expression of
The proteasome system
catalytic enzymes and their activation.
However, the susceptibility of proteins to degrada- The proteasome system is involved in protein loss in
tion may also depend on conformational stability of a synergistic way with the calpains. The proteasome
the protein, which is determined by factors such as is ubiquitous ATP- and ubiquitin-dependent proteo-
the intracellular temperature, free energy or pH. lytic system, which is able to degrade actin and
During muscle atrophy in response to disuse or myosin in vitro (Solomon & Goldberg, 1996). Pro-
immobilization, protein degradation is enhanced by teins to be degraded are modified by covalent con-
activation of proteolytic pathways (see for detailed jugation to multiple ubiquitin molecules, which
review, Jackman & Kandarian, 2004). Three intra- marks them for ATP-dependent degradation by the
cellular proteolytic systems are activated during proteasome complex (Ciechanover, 1994). During
muscle atrophy (Taillandier et al., 1996) and will be different conditions of disuse of mouse muscle, such
briefly discussed below: the calpain system, the as immobilization, denervation, hind limb suspen-
lysosomal system and the proteasome system. How- sion and fasting, the expression of the recently
ever, their relative contributions to the induction of identified muscle-specific F-box ubiquitine ligases,
muscle atrophy in different conditions remains to be MAFbx and MuRF1, was were enhanced (Bodine
elucidated by further research. et al., 2001a). For MAFbx, this was also the case in

360
Adaptation of muscle size and myofascial force transmission
fasting animals (Gomes et al., 2001). Inhibition of The endomysium is a tunnel-like structure that
one of these proteasome systems leads to substantial plays a major role in force transmission. A collection
attenuation of atrophy (Bodine et al., 2001a). Inter- of such tunnels, with shared walls between adjacent
estingly, recent data have shown that IGF-1 is a key tunnels, forms an integral part of the whole connec-
mediator of the expression of the MAFbx and tive tissue stroma of a muscle. For the elegant and
MuRF1 (Sandri et al., 2004; Stitt et al., 2004). thought provoking images of the microscopic struc-
IGF-1 activates the PI3K/AKT pathway, which in ture of muscle without muscle fibers see Trotter and
turn results in phosphorylation of the FOXO pro- Purslow (1992) and Nishimura et al. (1994).
teins, a subgroup of the Forkhead box O (foxo) Sarcomeres are connected via the cytoskeleton,
family of transcription factors. Phosporylation of trans-sarcolemmal molecules and laminin to the
the FOXO transcription factors results in their basal lamina. For a schematic view of molecules
cytoplasmic localization away from the target genes thought to be involved see Fig. 3. The basal lamina
in the nucleus (Brunet et al., 1999), which prevents is connected in turn to the endomysium probably via
transcription of the MAFbx and MuRF1 genes. proteoglycans (Nishimura et al., 1996). Therefore,
These findings indicate that IGF-1 is simultaneously the connective tissue stroma of a muscle (formed by
able to stimulate protein synthesis and suppress the endomysia, perimysia and epimysium as collagen
protein degradation. fiber reinforcement of the muscle ECM) is arranged
in series with the sarcomeres.
Other mechanisms: NO Work on force transmission within muscle with
non-spanning muscle fibers (i.e. muscle fibers that do
During remobilization of mice after hind limb un- not fully span the distance between the proximal and
loading, the soleus muscle shows sarcolemmal injury, distal aponeuroses, but end within the muscle belly)
which is accompanied by substantial increases in the has given another major push to the development of
number of neutrophils and macrophages (Nguyen & knowledge about mechanisms of force transmission
Tidball, 2003). As remarkably lower neutrophil con- by pathways other than myotendinous pathways
centrations were shown for muscles of transgenic (Loeb, 1984; Trotter, 1990; Hijikata et al., 1993;
mice, overexpressing nNOS during remobilization, it Trotter et al., 1995).
is suggested that NO helps to prevent enhanced For the particular case of non-spanning muscle
workload-related membrane damage (Nguyen & fibers, force transmission has been thought to occur
Tidball, 2003) and hence a breakdown of muscle between (parallel arranged) sarcomeres within neigh-
proteins. In addition, in myoblasts NO has been boring muscle fibers, and therefore those authors
shown to inhibit calpain-mediated proteolysis of (with the exception of Hijikata et al., 1993) have
talin and vinculin (Koh & Tidball, 2000). This referred to muscle containing non-spanning muscle
indicates that NO has the potential to increase fibers as series fibered muscle.
stability of cytoskeletal proteins and by doing so However, we have to consider the likeliness that
diminishes the rate of protein degradation by the most of the force is transmitted predominantly
proteasome system (see ‘‘The proteasome system’’). through the muscular connective tissue stroma rather
than onto the adjacent muscle fiber. In such a case
Essentials of myofascial force transmission non-spanning muscle fibers would be arranged in
parallel rather than in series. If shearing of the basal
Since 1983 (Street & Ramsey, 1965), it should have lamina and endomysial interfaces would not make
been clear to modern researchers that force transmis- the interface with the endomysium very stiff, very
sion pathways, in addition to myotendinous ones, are little of the force would be transmitted via such a
arranged in series with the sarcomeres within muscle pathway even in otherwise extreme conditions. In
fibers. Only in more recent years, evidence can be such conditions with tenotomy of a fraction of
found that a limited number of groups deal with this muscle fibers from a fully dissected muscle (prevent-
issue. If one reads much earlier literature thoroughly, ing myotendinous force transmission for the afflicted
it becomes clear that some people were aware of the muscle fibers), the force would be expected to fall
effects of such paths, even though they did not talk according to the decrease in physiological cross-
about it in the specific terms of force transmission. sectional area with tendinous connections at both
For example Kronecker and Cash (1880) showed ends of the muscle fibers. This was shown experi-
that muscles functioning within their natural con- mentally not to be the case (Huijing et al., 1998). In
nective tissue context are limited in their movement, addition, if the considerable force still exerted by the
compared with fully isolated muscle. Unfortunately, afflicted muscle fibers would have been transmitted
they decided that such constraints were not very onto the active sarcomeres of the neighboring muscle
important at sub-maximal levels of activation of fibers, having unaltered myotendinous function at
the muscle and left the idea alone. both fiber ends, the lengths of those sarcomeres and

361
Huijing & Jaspers
thus of those muscle fibers would have to be in-
creased substantially by the additional load imposed
on them. It was shown experimentally that this did
not occur (Huijing et al., 1998). Therefore, we con-
clude that most of the force that is transmitted by
shearing of the basal lamina and the interface be-
tween the groups of muscle fibers is transmitted
further via the muscular connective tissue stroma
and that is the reason why we should refer to the
whole process as myofascial force transmission.
The alternative correct way of expressing this is
that for a target sarcomere the myofascial connec-
tions form an additional load to be borne. Such an
additional load would prevent a sarcomere from
shortening sooner than in the case where only serial
sarcomeres loaded a specific target sarcomere.
Fig. 4. An example of proximo-distal force differences in rat
extensor digitorum longus (EDL) muscle. Force–time traces
Epimuscular myofascial force transmission of EDL isometric contractions are shown at two lengths. (1)
In almost all physiological experiments in vivo, in situ At low lengths EDL exerts a small force (a), but the
and in vitro, muscular force or its equivalent (e.g. amplitude of the distal force dominates that of the proximal
force. This occurs in both twitches seen at 200 and 400 ms, as
moment) is measured exclusively at one end of the well as during the plateau of the tetanic contraction. (2) At a
muscle (or at one joint). Implicitly, it is assumed that high length, obtained by proximal lengthening, EDL force is
the origin and insertion of the muscles (tendinous or much higher (b), but in this case the proximal force dom-
otherwise) are the only structures arranged in series inates the distal force, i.e. the side of dominance of force is
with the sarcomeres of the muscle: in such a case reversed.
proximally and distally exerted forces would be
identical. The arguments presented above have led to surrounding tissues. Proximal or distal lengthen-
us to initiate simultaneous measurement of force at ing to the same muscle length yielded quite different
proximal and distal tendons of muscle, where possi- effects on the forces exerted at proximal and distal
ble. Results of those experiments have led to a major tendons, as well as on the proximo-distal force
change in our views on muscular function. The main difference (Huijing & Baan, 2003). For distal length-
visions of muscular function derived from experi- ening, distal force increased more than proximal
mental as well as modelling results regarding such force after proximal lengthening of EDL. The sign
conditions will be presented below. of the proximo-distal force difference reversed for
If force is transmitted from the muscular stroma to proximal compared with distal lengthening. Also, for
anything else other than the muscles own origin or distal lengthening, the proximo-distal force differ-
insertion tendons, we speak of epimuscular myofas- ences were higher.
cial force transmission. An alternative way to express The specific effects of relative position can be
the same condition is to say that the connective shown more clearly if a muscle at constant length is
tissues outside the muscle of interest may constitute moved through its connective tissue context (Maas et
an additional load on all or a fraction of the al., 2004): depending on position, the proximo-distal
sarcomeres within the muscle fibers. force difference changed not only in magnitude, but
also in sign!
Characteristic feature: proximo-distal force differences
Two types of epimuscular transmission
In all conditions with any net epimuscular force
transmission, forces exerted at origin and insertion Two types of epimuscular myofascial force transmis-
of a muscle or a muscle fiber are not identical sion are distinguished:
(Huijing & Baan, 2001a). The additional load im-
posed by epimuscular connective tissue structures Intermuscular myofascial force transmission. If the
will affect forces exerted at specific tendons. Figure myofascial load on the sarcomeres within the fibers
4 shows an example of such force difference for a of a muscle is imposed via the direct and very short
tetanic contraction of rat EDL muscle that is active connections of two adjacent muscular connective
within its natural connective tissue context. It should tissue stromata, we speak of intermuscular myofas-
be noted that an extremely important variable, in cial force transmission. In such a case shearing of the
addition to muscle length, is muscle position relative shared epimysium may yield the stiffness that allows

362
Adaptation of muscle size and myofascial force transmission
cial force transmission between sarcomeres of a
muscle fiber and the extramuscular structures.
A major extramuscular structure that may be
involved in this pathway is the neurovascular tract,
i.e. the collagen fiber reinforced sheet or bundle of
connective tissues that envelops and protects blood
vessels, lymph vessels and nerves and their braches
outside the muscle (Fig. 5). The sheet or bundle is
Fig. 5. The neurovascular tract as seen in a lateral view of continuous with the muscular stroma along most of
the dissected rat lower leg. If one loads a dissected extensor
digitorum longus (EDL) at proximal and distal tendons (i.e.
the length of the muscle belly, as the continuously
in vertical direction of the image), as the EDL is pulled branching nerves, blood and lymph vessels within the
down, a connective tissue sheet is exposed that connects the muscle are embedded within the peri- and endomy-
intramuscular connective tissue stroma of the EDL (and the sia. However, at specific locations, major branches of
other muscles, not shown) to other passive elements of the blood vessels and nerves enter the muscle belly. At
anterior crural compartment. As this sheet envelops both
nerves and blood vessels that either enter the muscles of the
the other extremity of the sheet, the neurovascular
anterior crural compartment or continue on into the foot or tract may also be attached to structures (i.e. inter-
into the peroneal compartment we refer to it as neurovas- muscular septa, interosseal membrane and periost)
cular tract. It should be noted that in vivo the neurovascular forming the walls of a compartment in which a
tract does not necessarily have the shape of a sheet. Con- muscle group is organized. It should be realized
nections of this tract are made along its full length to the
interosseal membrane, the anterior intermuscular septum
that such structures are, sometimes very directly,
and the periost of the tibia. The tract also forms a connec- connected to the capsule and ligaments of the joint
tion to the peroneal compartment and its muscles as it passes (Fig. 6), so that some of the extramuscularly trans-
through a fenestra (or window) in the intermuscular septum mitted force may be used for stabilization of the
(see also Fig. 6). The smallest divisions on the ruler indicate joint.
millimetres.
In most physiological experiments on muscle, the
muscles are fully dissected from their surroundings
force transmission. If such intermuscular connec- with the exception of their innervation and blood
tions are stiff enough, force that has its origin within supply, the integrity of which is usually crucial for
sarcomeres of one muscle may be exerted via the the experiment. This means that in the so-called
direct stroma–tendon connections at the tendon of in situ experiments at least a remnant of the neuro-
an adjacent muscle. vascular tract remains. Such a remnant was shown
to be capable of maintaining a substantial proximo-
Extramuscular myofascial force transmission. If the distal force difference for passive, but not for active
myofascial load on the sarcomeres within the fibers force (Huijing & Baan, 2001a). However, it has since
of a muscle is imposed via non-muscular connective been shown that the existence of a proximo-distal
tissue elements, we speak of extramuscular myofas- active force difference is dependent on the relative

Fig. 6. Connections of passive elements of the anterior crural compartment to ligaments of the knee, in addition to the general
fascia. The major connective tissue structure shown in this image of the fully dissected rat anterior crural compartment is the
anterior intermuscular septum (SIA) with its fenestra (F). The SIA is being strained via the forceps that is pulling on dissection
remnants of the general fascia seen in the bottom. SIA is attached at almost right angles to the interosseal membrane, which
runs between the tibia (T) and the fibula below the tibia in this image (not to be seen). Note that at the bottom left the SIA is
supported by the lateral collateral ligament that crosses the knee joint. The tibia is surrounded by its own membrane, the
periost (p), which is also connected to the medial collateral ligament (med CL) of the knee. Note that a part of any force exerted
via the neurovascular tract onto these structures may be exerted at the knee.

363
Huijing & Jaspers
position of the ‘‘fully dissected muscle’’ with respect forces to its outside. The load imposed on the
to the neurovascular tract and other tissues (Rijke- sarcomere by the origin and insertion will definitely
lijkhuizen et al., 2004; Yucesoy et al., 2005). act as such an opposing force. However, the epimus-
It should also be noted that the neurovascular tract cular load is, like the myotendinous load, arranged in
forms an indirect connection between the stromata of series with the sarcomere and thus will contribute to
two adjacent (i.e. synergistic) muscles. Similarly, the force that will equilibrate with the force exerted
neurovascular tracts even form a connection between by the contracting sarcomere.
bellies of (antagonistic) muscles located in adjacent It should be noted that the stiffer parts of the
compartments. neurovascular tract (i.e. the locations where the
Recent evidence indicates that active force may nerves and blood vessels do enter the muscle) are
even be transmitted via extramuscular myofascial not distributed uniformly over the muscle belly, but
pathways to the insertion of a muscle on bone via are found at specific locations. As a consequence, the
epitendinous connective tissues (Rijkelijkhuizen et al., epimuscular load is distributed over the muscle
2004). stroma, and the loads will not be distributed uni-
Recently, for some human cadavers, Bojsen-Møl- formly to all sarcomeres within the fibers of the
ler et al. (2004) reported a rare anatomical variation muscle. Therefore, the sarcomeres exposed to a lower
consisting of aponeurotic connections between distal load will shorten more before isometric force equili-
aponeuroses of gastrocnemius and soleus muscle. brium can be reached than the sarcomeres that are
Similar connections are also known between some more heavily loaded.
of the distal tendons of human extensor digitorum
communis muscle, but they seem to occur more
frequently there. Even though these connections Force transmission between antagonistic muscles
can yield mechanical interaction between the con-
nected muscles, they should not be confused with The question posedis whether such intra-compart-
myofascial connections, since the connections occur mental effects of interaction between muscles will
along the myotendinous rather than the myofascial also be present for muscles located within different
paths. compartments (Huijing, 2003). Potential myofascial
connections between antagonistic muscles are per
Epimuscular myofascial force transmission between definition only of an extramuscular nature, because
adjacent synergists there is no direct contact between the stromata of
antagonist muscles. This illustrates that intermus-
For conditions involving several muscle groups acti-
cular myofascial effects can in principle also be
vated and kept at constant muscle tendon complex
mediated by extramuscular tissues.
lengths, lengthening of one muscle or complex of
The reasoning behind such hypotheses regarding
muscles affects force exerted at their origin and
myofascial interaction between antagonistic muscles
insertion by adjacent muscles within the compart-
is based on the fact that the relative positions of
ment. This is a rather general feature (Maas et al.,
antagonistic muscles change most drastically of all.
2001, 2003a; Huijing, 2002; Huijing & Baan, 2003;
If extramuscular myofascial connections of sufficient
Huijing et al., 2003; Yucesoy et al., 2003). Figure
stiffness exist between antagonistic muscles, we
7a, b shows an example of such results. In that case,
should expect very substantial effects. Figure 7a, c
length–force characteristics of the tied rat tibialis
shows preliminary experimental results indicating
anterior and extensor hallucis longus complex were
that this is indeed the case. As force exerted by the
determined. Length and position changes of that
rat tibialis anterior and extensor hallucis longus
complex caused substantial changes in the proximo-
muscles at their tied together distal tendons was
distal active force difference of EDL, which was
determined at progressively higher lengths, distal
kept at constant muscle–tendon complex length.
force exerted by the antagonistic peroneal group
This indicates changes in epimuscular myofascial
dropped progressively to as much as 25% of the
force transmission between EDL and surrounding
initial force, despite the fact that the muscle tendon
structures within the anterior crural compartment.
complexes of the peroneal group were kept isome-
trically.
Substantial serial distribution of sarcomere length expected
It should be noted that during in vivo movement
Intuitive reasoning leads to an expectation of serial the relative positions of the two muscle groups
sarcomere length distributions. Any active sarcomere changes even more than in the experiment performed
will shorten to its active slack length if unopposed by because both synergistic and antagonistic muscle will
an external force (load) that balances the sarcomere change their length in opposite directions, so that
force. The active slack length is defined as the length at the extremes of the movement range one should
at which the sarcomere is active, but cannot exert expect very sizable effects.

364
Adaptation of muscle size and myofascial force transmission
muscles and tendons we should consider exclusively
not only the condition of the target muscles, but also
the conditions of all other muscles within the same
segment. Since several muscles related to a particular
segment are bi- or even poly-articular, this also
means that the conditions at joints further removed
from the segment of interest should be taken into
account when specifying experimental conditions of
a specific target muscle regarding muscular adapta-
tion and its effects.
It may be this factor that has added to the difficulty
of interpreting mechanisms of adaptation active in
classical experiments. Therefore, it is concluded that
the usual classical experimental conditions are too
complex to permit an enhanced understanding of the
real determining factors, let alone the mechanisms of
adaptation. It is clear that if we want to attain goals
described in the introduction a new approach to the
study of muscular adaptation of serial sarcomere

Fig. 7. Extramuscular myofascial force transmission be-


tween synergistic and antagonistic muscles: (a) length force
characteristics (active and passive) of the rat tibialis anterior
and extensor hallucis longus muscles of which the distal
tendons have been tied together (TA1EHL). Note that the
length of this complex was exclusively altered in this experi-
ment. (b) The proximo-distal force difference of rat EDL
(synergist of TA and EHL) and the effects of changing
TA1EHL length on it. Note that as TA1EHL is active at
higher lengths, the proximo-distal force difference first
increases its negative amplitude and then decreases it, despite
the fact that EDL muscle–tendon complex length was un- Fig. 8. Combined experimental and finite-element model
changed. (c) Force exerted by all peroneal muscles (PER), results for distal lengthening of rat extensor digitorum
while being kept at constant muscle-tendon complex length, longus (EDL) with epimuscular myofascial connections.
at different lengths of antagonistic TA1EHL. Force is The top panel shows experimental length–force curves of
expressed as percentage of initial force (at low TA1EHL rat EDL muscle, as well as a modelled segment of that curve.
length). Note that as TA1EHL length and active force Distal lengthening was used to change the EDL length. EDL
increase, PER active force decreases by as much as 30% length is expressed as a deviation (Dlm1t dist) from initial
(use left Y-axis), despite being kept at constant length. A low length. Force was normalized for optimal distal EDL
similar feature is seen for EDL distal active force, but the force (Fmao EDLdist). In the specific conditions of these
amplitude of that decrease is limited to about 6% (use right experiments, at all modelled lengths, distal force (dist)
Y-axis in grey). TA1EHL length is expressed as a deviation dominates proximal force (prox). Solid markers indicate
from its optimum length and the X-axis applies to all plots of model force. For the length indicated (arrow), the lower
this figure. panel presents the distribution of strain in the muscle fiber
direction (indicated by a solid line with marking for prox-
imal fiber end (prox) and distal fiber end (dist)). The strain
value represents the fractional change from the initial length
In several different experiments in our laboratory (e.g. 0.15 represents a 15% shortening with respect to
(unpublished observations), we have found similar initial conditions). Note the particularly high serial distribu-
effects and interactions for all antagonistic muscle tion of strains within the model, indicating a high serial
groups within the rat lower part of the hind limb. distribution of sarcomere lengths within muscle fibers. Note
From such results, we conclude that even antagonis- also that the lower proximal force is related to shortened
sarcomeres, not exposed to the added epimuscular myofas-
tic muscles should not be considered as being in- cial load that allows distal sarcomeres within the same
dependent of each other and that by specifying muscle fibers to be at higher lengths. The dotted contour
experimental conditions regarding adaptation of indicates the starting condition of the muscle.

365
Huijing & Jaspers
number and physiological cross-sectional area must muscle during the classical experiments on adapta-
be developed. tion are actually very complex, because the condi-
tions even differ locally along the length of one
muscle fiber.
Finite-element models of epimuscular myofascial force It should be noted that, for in vivo magnetic
transmission confirm relatively high serial and parallel resonance imaging (MRI) measurement of strain
distribution of muscle fiber strain and displacement velocity of aponeurosis and mus-
Since during myofascial experiments, the connective cular elements within human muscle, considerable
tissue around the muscle belly was left intact as much distributions of the values of these variables are
as possible we cannot study details of the muscle, reported (Pappas et al., 2002; Finni et al., 2003a, b)
because we cannot visualize them. Finite-element within a muscle. Such results are very difficult to
models have helped us to understand what happens explain without the concepts of epimuscular myo-
within the muscle in conditions where epimuscular fascial force transmission. In any case they at least
myofascial force transmission is evident from simul- confirm the in vivo presence of the types of distribu-
taneous force measurements at both proximal and tions foreseen on the basis of the finite-element
distal tendons. Figure 8 shows an example of com- model calculations. More specific modelling of these
bined experimental and model results. For details of in vivo results is indicated to aid their interpretation.
such modelling and experiments, see Yucesoy et al.
(2003). A clear proximo-distal total force difference is
present in rat EDL muscle at a length over optimum Different species and epimuscular myofascial force
length. Calculated local muscle fiber strains (repre- transmission
sented as the fraction of actual lengths over the initial Epimuscular myofascial force transmission has been
lengths) yield an estimate of local sarcomere lengths. shown to exist and have substantial effects in mam-
Despite the high length of the muscle, particularly at malian muscle both in rodents Huijing, 1999, 2002;
the extramuscularly connected face of the model, Huijing & Baan, 2001b, 2003; Maas et al., 2001,
sarcomeres located near the proximal end of muscle 2003a–c, 2004; Smeulders et al., 2002; Huijing et al.,
fibers are shortened by up to 14% below their initial 2003; Yucesoy et al., 2003; Rijkelijkhuizen et al.,
length, whereas sarcomeres closer to the other (dis- 2004) and in human patients suffering from spastic
tal) end of the same muscle fiber are lengthened by up paresis (Smeulders et al., 2002, 2003, 2004a, b, c;
to 50%. The distal sarcomeres are lengthened much Kreulen et al., 2003).
more because extra- and intermuscular myofascial Below we will present evidence regarding adapta-
forces load them additionally. It is clear that such tion in amphibian muscle fibers. Therefore, it is
myofascial connections cause high and stable serial necessary to ascertain that perimuscular myofascial
distributions of sarcomeres, which lead to the exer- force transmission is also active in these species.
tion of different active and passive forces at the Presently, we are only able to present some prelimin-
proximal and distal end of each muscle fiber. ary data on Xenopus laevis muscle. However, such
Such phenomena have very many functional con- data are essential for the line of reasoning of this
sequences. For example, intersarcomere dynamics article.
are not exclusively interactions between only sarco-
meres in series, as always assumed in the literature
Summary of methods used in X. laevis force transmission
(e.g. Denoth et al., 2002; Telley et al., 2003), but
experiments
actually constitute interactions between new units
entities arranged in series. This new unit is consti- The experimental approach was similar to that used
tuted by a sarcomere together with its relevant part for rats: the animal was anaesthetized using urethane
of the endomysium of the muscle fiber (arranged in solution (dose 0.05 mL/g body mass of 12.4% solu-
parallel to the specific sarcomere). Serial interaction tion). Over the target muscle, skin and subcutaneous
between such new units is much more stable me- tissues were removed and fasciotomy was performed
chanically because of the parallel paths of force on the anterior tibial compartment. A muscle with
transmission. Therefore, myofascial force transmis- both proximal and distal tendons was selected: in this
sion is very likely to prevent the so-called popping case TA muscle with one tendon crossing the knee
sarcomeres, which have been presumed to occur joint and two distal tendons crossing the ankle joint.
(Morgan, 1990) because only the sarcomeres were A force transducer was attached to both the proximal
considered as exclusive serial units. tendon and the combined distal tendons. The ankle
Also for adaptation, this finding is highly relevant, was maximally plantar flexed to allow free passage of
because conditions will be very different for different the distal TA tendons and their connections. High in
parts of the same muscle fiber. This means that the the femoral compartment, the distal end of the cut
ostensibly relatively simple conditions imposed on a sciatic nerve was stimulated maximally at 80 Hz: all

366
Adaptation of muscle size and myofascial force transmission
cle fibers is dependent not only on the length of the
muscle fibers, but also on the relative position of
those fibers with respect to other muscles and con-
nective tissues within the limb.
Direct intervention with the connective tissues, as
in ablation of synergistic muscles, will change the
conditions of myofascial force transmission drasti-
cally. Simple fasciotomy of the compartment as well
as progressive dissection of muscles and tendon
already does that (Huijing & Baan, 2001a; Smeulders
et al., 2002; Huijing et al., 2003; Kreulen et al., 2003;
Rijkelijkhuizen et al., 2004). Release of a retinaculum
does not only change the length and length range of a
muscle of which tendons pass through but it will also
change the relative position of the muscle with
Fig. 9. An example of length–force characteristics of Xeno-
pus laevis tibialis anticus longus muscle embedded within its respect to connective tissues of a compartment as
natiural connective tissue context. Active force exerted by well as to other synergistic and antagonistic muscles.
maximally active TA was measured at its proximal tendon, Simple tenotomy will not remove a muscle from
as well as the summed forces exerted by its two heads at the functioning because of myofascial force transmis-
distal two TA tendons that were connected to one force sion, because the muscle will not shorten to its active
transducer. Note that the proximo-distal active force differ-
ences observed are particularly high at low lengths, with slack length (Kreulen et al., 2003), but will be
proximal force dominating distal force. At length higher restrained at higher lengths and may even be length-
than the crossover point (approximately Dl 5 5 mm), distal ened by surrounding tissues.
force dominates proximal force but with smaller differences. The serials distribution of sarcomere length within
The presence of such force differences indicates epimuscular muscle fibers, enhanced by myofascial effects, raises
myofascial force transmission.
doubt on the concept, introduced above, that the
condition of the muscle fiber as a whole will be
muscles in the lower leg were simultaneously active the determining factor of adaptation. Considering
maximally. Ambient temperature was regulated at the asymmetries in muscle length effects (e.g. much
22 1C. more powerful adaptation effects of high length
compared with low lengths: Williams, 1990), adapta-
Preliminary results tion of muscle fiber size may be a much more local
affair involving only parts of the muscle fiber. It is
At all muscle–tendon complex lengths studied, but conceivable that, regarding adaptation, the muscle
one, a proximo-distal force difference was found: at fiber as a syncytium should not be considered as one
low lengths considerable distal active force was unit, but rather as a collection of units of adaptation,
exerted (420% of distal optimal force), but simulta- each of which react to its own local-specific mechan-
neously proximal active force equalled zero (Fig. 9). ical stimuli.
At progressively higher lengths obtained by distal Therefore, it is also possible that the specific
lengthening, proximal as well as active distal force myofascial effects on a muscle fiber will be respon-
increased, but not in a similar fashion: distal active sible for signals of adaptation. It is clear that to be
force increased much more. As a consequence, force able to get closer look at the detailed mechanisms of
values approached each other, and after further muscular adaptation we need to create experimen-
lengthening the proximo-distal force difference re- tally mechanical conditions which are less com-
versed its sign, i.e. distal force became dominant over plex than those usually afforded during in vivo
proximal force. These results are very similar to those experiments.
of previous experiments in other species and indicate
that epimuscular myofascial force transmission is
also active in X. laevis muscle.
Study of adaptation at the cellular level
Unravelling the mechanisms of adaptation of muscle
Some important consequences of myofascial force size requires a sophisticated in vitro approach to
transmission for the study of adaptation overcome the problems related to the substantial
The conditions imposed on the muscle fibers of a distributions of sarcomere strains expected within
muscle that is working within the natural context of muscle and muscle fibers during in vivo conditions.
its epimuscular connective tissues are quite complex. Several in vitro setups have been developed for
The serial sarcomere length distribution within mus- mature mammalian, single muscle fibers. Since their

367
Huijing & Jaspers
initiation in the forties of the last century, such via the cytoskeleton onto the myonucleus and leads
systems have primarily been used for acute investiga- more directly to gene expression.
tion of physiological and mechanical properties of
amphibian muscle fiber (e.g. Ramsey, 1947; Natori,
Mechano-chemical transduction
1954; Gordon et al., 1966; Lannergren, 1978; Wes-
terblad & Lannergren, 1986; Van der Laarse et al., Within cell membranes, several trans-membrane re-
1991). ceptors and channels are recognized that are able to
However, more recently they have also been used elicit biochemical activities upon mechanical loading.
to study acute effects of hormones and growth Within muscle, the trans-sarcolemmal proteins most
factors on cytoplasmic [Ca21] (Bruton et al., 1999). likely involved in the adaptation of muscle size are
In addition, long-term cultures of single mature the integrins, the dystroglycans and the stretch-acti-
muscle fibers have also been developed and used to vated calcium channels (for reviews, see Carson &
study [Ca21] homeostasis (De Backer et al., 2002), Wei, 2000; Ruwhof & Van der Laarse, 2000; Rando,
the effects of growth factors on satellite cell prolif- 2001). Among these, the integrins and dystroglycans
eration (Bischoff, 1986) as well as MHC gene expres- are the core proteins of adhesion complexes that
sion in response to different activation regimes (Liu connect the ECM and the cellular cytoskeleton.
& Schneider, 1998). Such in vitro systems, however, Pardo et al. (1983) first recognized the connection
are inappropriate for the investigation of long-term of the myofibrils to the sarcolemma. Because of their
adaptation of muscle fiber size, because muscle fibers shape of the immunofluorescence they called these
are maintained in the presence of serum and fiber sites ‘‘costameres’’. Later, the costameres were shown
length cannot be manipulated as the muscle fibers are to be sites of transmission of myofibrillar force of
released from the tendons during the isolation by adult cardiac myocytes to the ECM (Danowski et al.,
using catalytic enzymes that facilitate cell isolation 1992) and were hypothesized to reinforce and stabi-
(Bischoff, 1986; Liu & Schneider, 1998). In addition, lize the sarcolemma in skeletal muscle fibers (Petrof
these enzymes may cause damage to or removal of et al., 1993). Subsequently, these trans-sarcolemmal
the endomysium (Bischoff, 1986) and basal lamina, complexes were shown to be also involved in activa-
as possibly was the case with Liu and Schneider tion biochemical-signalling pathways.
(1998), which may affect both the ligand-receptor
signalling and the mechanical signalling via trans- The integrins
sarcolemmal complexes.
The integrin family can be described as heterodimeric
Most of our knowledge, regarding (1) how me-
transmembrane glycoproteins consisting of a and b
chanical signals affect cells and (2) how the signals
subunits. Binding the integrin to glycoproteins in the
are subsequently converted into intracellular bio-
ECM, such as fibronectin and laminin, results in
chemical activities that regulate protein synthesis
the formation of sub-sarcolemmal FACs within the
and degradation, was based, until recently, on results
muscle fibers, in which vinculin is thought to provide
of culture experiments using myoblasts or non-mus-
a major mechanical linkage (Fig. 3) between the
cle cells such as epithelial cells, fibroblasts or osteo-
integrin and the subsarcolemmal actin filaments
blasts. These types of cells are morphologically and
(different from sarcomeric actin filaments), which
functionally different from mature muscle fibers
are part of the cytoskeleton (Berthier & Blaineau,
and, generally, culture of these types of cells needs
1997). Also bound to vinculin is talin (Fig. 3).
to be performed in the presence of a serum, of
Loading of FACs stimulates translocation and accu-
unspecified content, to maintain the cells viability.
mulation at these sites of kinases, small GTPases
Despite the limitations of these models, such experi-
and other signalling molecules (e.g. PI3K, FAK and
ments have substantially enhanced our understand-
Rho) (for reviews, see Ingber, 1997; Janmey, 1998;
ing of fundamental mechanisms of cellular signal
Carson & Wei, 2000). These signalling molecules are
transduction.
able to activate several signal transduction pathways
Two pathways of mechano-transduction are distin-
such as the MAPK and the PI3K–mTOR pathway,
guished: (1) mechano-chemical signal transduction to
which in turn increase both mRNA expression and
the nuclei, i.e. an indirect pathway by which mechan-
translation (see ‘‘Biochemical-signalling pathways’’).
ical stress applied to the muscle–tendon complex is
transmitted onto the ECM including the endomysium
The dystroglycan complexes
to trans-sarcolemmal structures of muscle fiber and
subsequently converted at that location into biochem- In addition to the integrins, the dystroglycans also
ical signals as treated above and (2) mechano-trans- provide a mechanical linkage between the ECM and
duction, i.e. pathways by which the mechanical load the cytoskeleton (Berthier & Blaineau, 1997; Rando,
exerted on the endomysium–basal lamina–trans-sar- 2001). Whereas vinculin is indicated as the major
colemmal complexes of the muscle fiber is transmitted protein that connects the integrin to the cytoskeleton,

368
Adaptation of muscle size and myofascial force transmission
for the dystroglycans, dystrophin is identified to form connection to the cytoskeleton (i.e. the spectrin
the connection to the cytoskeleton. Like for the system, Berthier & Blaineau, 1997).
integrins, the dystroglycan complexes are also assem-
bly points for signalling molecules such as FAK and
RhoA, which have been shown to be able to mediate Mechano-transduction
biochemical-signalling pathways affecting the rate of The alternative way by which mechanical load ex-
protein synthesis (Berthier & Blaineau, 1997; Rando, erted on cells is transduced into changed protein
2001). synthesis or degradation is based on the concept
Evidence of the involvement of the dystroglycan that cells are hard-wired systems according to the
complexes in mediating biochemical-signalling path- principles of tensegrity. Combinations of stiff rods
ways such as the MAPK, the NO and the PI3K– and pre-stressed elastic strings characterize tensegrity
mTOR pathways is emerging (Rando, 2001; Langen- structures that yield stability. Movement of one
bach & Rando, 2002; Spence et al., 2004). The element of a tensegrity system involves movement
finding that dystrophin knockout mice do not ex- of the integral structure. Ingber (1997, 2003a, b) has
press MGF (Goldspink et al., 1996) also indicates postulated the model of tensegrity-based signalling in
involvement of the dystroglycan system in the induc- biological cells. This model proposes that the cytos-
tion of muscle hypertrophy. MGF is a potent sti- keleton inclusive costameres constitute a pre-stressed
mulator of proliferation of myoblasts (Yang & Gold- tensegrity structure that allows force transmission
spink, 2002) and therefore is likely to play an from the collagen fiber reinforced ECM onto the
important role in the activation of satellite cells. cytoskeleton and nucleus (Wang et al., 1993; Ingber,
The precise mechanisms of dystroglycan complexes 1997). Force exerted via the ECM will have two
involvement in adaptation of muscle size remain to effects:
be determined. (a) Release of mRNA and ribosomes attached to the
cytoskeleton and translocation to the sites, where
Linking of the integrin and the dystroglycan systems
protein synthesis is required (Chicurel et al., 1998).
As dystrophin and talin bind with high affinity (b) Nuclear deformations or conformational changes
(Senter et al., 1993; Yoshida et al., 1998), dystrophin of the chromatin that directly may affect the trans-
also plays a role in linking both costameric trans- criptional activity (Bloom et al., 1996).
sarcolemmal systems. Consequences of connecting
Accumulating evidence supporting this model in
these two systems for force transmission and the
the regulation of cellular organization is largely
pathology of Duchenne muscular dystrophy (i.e.
based non-muscle cells (Chicurel et al., 1998; Lelievre
dystrophin deficiency) have not been considered
et al., 1998; Thomas et al., 2002).
as yet. In contrast, regarding signal transduction bi-
With respect to adaptation of skeletal muscle, the
directional communication between the dystrophin-
involvement of mechano-transduction in the regula-
containing complex and the integrin adhesion system
tion of adaptation of muscle size remains to be
has been shown in cultured myocytes (Yoshida et al.,
characterized. For lengthening of cardiomyocytes,
1998). Further work on this topic is indicated.
it was shown that cytoskeleton desmin filaments
transmit force onto the nucleus (Bloom et al.,
Stretch-activated channels
1996). It was hypothesized that this could alter the
Another type of mechano-sensitive trans-sarcolem- conformation of the chromatin and possibly stimu-
mal structure is the stretch-activated channel. Stretch late transcription. It should be realized that tensegr-
of cardiac myocytes causes direct influx Ca21 via ity effects have only been shown for relatively small
these channels (Gannier et al., 1996; Tavi et al., cells (diameter in all directions o20 mm). Since
1998), which likely activates the Ca21-mediated muscle fibers are very big multinucleated syncytia
pathways as mentioned above. (for example adult humans: cross-fiber diameter 450
At higher lengths, mature isolated frog muscle mm, 1 cmolengtho30 cm), it is very well conceivable
fibers show a length-dependent increase in free in- that the muscle fiber consists of many units of
tracellular [Ca21], starting at a mean sarcomere tensegrity regulating muscle fiber size, i.e. the adap-
length of 2.4 mm (Snowdowne, 1986). Stretch-acti- tations taking place within these local units.
vated channels are also detected in skeletal muscle In addition, changes in nuclear shape may also
fibers (McBride et al., 2000) and may mediate eleva- cause entry of [Ca21] into the myonucleus as was
tion of intracellular [Ca21]. Whether the stretch- shown in isolated nuclei in response to osmo-me-
activated channels are involved in the induction of chanical stress (Itano et al., 2003).
skeletal muscle hypertrophy remains to be clarified. However, direct mechanical signalling via the
With respect to this, it should be noted that the cytoskeletal molecule desmin was not confirmed by
stretch-activated channels colocalize with a system of experiments with desmin knockout mice. After im-

369
Huijing & Jaspers
regulating adaptation of muscle fiber size indepen-
dently, as well as allow study of their interactions.
A unique culture system for mature single Xenopus
muscle fibers was developed by Lee-De Groot and
Van der Laarse (1996), which meets most of these
requirements. Using this system, these authors
showed that it is feasible to culture mature single
muscle fibers for 2 weeks. However, when using a
serum-free culture medium, as culture time pro-
gressed twitch force declined and the muscle fibers
were shown to be metabolically unstable (Lee-de
Groot & Van der Laarse, 1996).
However, the feasibility of dissections of single
muscle fibers with intact basal lamina and endomy-
sium and their culture warranted further develop-
Fig. 10. Serial sarcomere length distribution in cultured ment of a system with the use of which, mature,
single Xenopus laevis muscle fibers. Using laser diffraction single Xenopus muscle fibers could be cultured in
techniques, saromere length was measured at intervals along
the length of the muscle fiber-basal lamina-endomysium stable conditions for up to months.
units cultured successfully for several weeks. Position along In close collaboration between the laboratories of
the fiber is quantified as deviation from the proximal Huijing and Van der Laarse, Jaspers developed a
myotendinous junction and normlized for muscle fiber new culture medium and tuned the system such that
length. Note the rather limited size and location of serial muscle fibers can be maintained metabolically stable,
distribution of sarcomere length. Note also that these values
are very much lower than those expected of the basis of while tetanic force as well as adaptation of the serial
finite-element modelling for fibers within a muscle exposed sarcomere number can be studied longitudinally. The
to epimuscular myofascial force transmission. success of this new, well-defined culture medium was
proved, as we were able to culture single mature X.
mobilization of mice muscles at high or low length, laevis muscle fibers for up to three months, while
desmin knockout mice showed the same adaptations tetanic force was stable (Jaspers et al., 2001; Jaspers
of the muscle physiological cross-sectional area, as et al., 2004).
well as the serial sarcomere number as for wild types
(Shah et al., 2002). This may indicate that mechano-
transduction via desmin is either not relevant in the
regulation of muscle size, or, alternatively, that other Dissection and culturing of single Xenopus muscle fibers
cytoskeletal proteins replacing desmin or acting in- Dissection of single Xenopus muscle fibers from small
dependently are involved in mechano-transduction fascicles attached to remnants of tendon requires
as well. special skills. In Xenopus muscle this is generally
somewhat easier than in mammalian muscle, be-
cause, in contrast to mammalian experimental ani-
First attempts at a new approach: long-term culture of mals, the dimensions of the muscle fibers (diameter
mature, single muscle fibers up to 100 mm) are similar to those of muscle fibers of
adult humans. Measurement of the relationship
Because of considerations of myofascial force trans- between muscle fiber cross-sectional area and the
mission, an experimental setup for single muscle oxidative capacity of muscle fibers has shown that
fibers from adult animals in vitro is required that the metabolic fluxes of muscle fibers from X. laevis
allows long-term culture of muscle fibers with intact and humans are similar, whereas those of rodent
basal lamina and endomysium. In fact it should be muscle fibers are substantially higher (Van der
feared that any method of isolation of muscle fibers Laarse et al., 1991). A drawback may be that the
that would damage the basal lamina–endomysium myonuclei of Xenopus are, in contrast with human
complex would lead to substantial changes of muscle fibers, not located exclusively at the periphery
function of the muscle fiber and affect viability in a of the fiber.
major way. Single muscle fibers have been isolated aseptically
Using a culture setup as indicated, factors such as from fascicles of the adult X. laevis iliofibularis
global or mean muscle fiber strain, hormonal com- muscle by cutting at the endomysia of surrounding
position of the culture medium as well as the type muscle fibers and removing the muscle fibers sur-
and degree of contractile activity can be manipulated rounding a target fiber. Such dissection leaves the
independently during the culture. Such a system endomysium and basal lamina surrounding the tar-
should allow investigation of the effects of the factors get muscle fiber (see Fig. 9 of Jaspers et al., 2004).

370
Adaptation of muscle size and myofascial force transmission
In addition to the high control of the physiological ever, acutely after the culture, the increase in tetanic
conditions (overall strain, serum-free medium; for force could be made to disappear immediately after
details see Jaspers et al., 2001, 2004), a very impor- the muscle fiber had been activated tetanically at a
tant methodological advantage is that the parameters high length (i.e. over a mean sarcomere length of 2.3
of interest can be studied longitudinally, which elim- mm) and tetanic force was again similar to its initial
inates the need for group statistics and avoids diffi- value before culture. This is a relatively ill under-
cult decisions regarding control groups. stood phenomenon requiring further research.
Using such methods, not only adaptation at the It is concluded that culture muscle fibers at differ-
supra-molecular level but, using in situ hybridization, ent overall strains neither induce atrophy or hyper-
also the very early signs of preparation for adapta- trophy, nor a major change in the serial sarcomere
tion can be studied(i.e. mRNA transcription) (Jas- number. Therefore, it is concluded that in vitro
pers, 2002; Jaspers et al., 2002). adaptation of Af and serial sarcomere number is
not regulated by overall or local muscle fiber strain
per se.
Overall strain does not uniquely regulate muscle
fiber size
Using a new culture medium, we investigated the Discrepancy between adaptation effects in vivo and
effects of culture of the muscle fiber at a mean in vitro
sarcomere length of 2.3 mm (l2.3 mm). At this length, Comparison of such in vitro results with those of the
the muscle fiber is just over its passive slack length. classical experiments in vivo reveals strikingly differ-
Before and after culture, the coefficient of variation ent effects in response to long-term maintenance of
in sarcomere lengths along the muscle fiber muscle fibers at high or low lengths. Because of the
amounted to less than 2% (Fig. 10). Note that the isolation of the muscle fibers and the culture itself,
value is very much lower than those obtained for the physical as well as the biochemical environment
muscle fibers during epimuscular myofascial force of the muscle fiber hasbeen altered compared with
transmission as predicted by finite-element modelling the in vivo situation. The discrepancy reported yields
(see ‘‘Finite-element models of epimuscular myofas- challenges to identify mechanisms that are active
cial force transmission confirm relatively high serial during adaptation in vivo, but which may not be
and parallel distribution of muscle fiber strain’’). active in vitro and vice versa.
During culture of up to 2 weeks at l2.3 mm, tetanic
force was shown to remain constant and the serial
Molecular factors
sarcomere number was not changed (Jaspers et al.,
2001). These data indicate similar rates of protein The presence of autocrine/paracrine growth factors
degradation and synthesis during culture at this fiber at the sarcolemma, in combination of high overall
length. strain, is usually required for induction of hypertro-
In order to test whether overall fiber strain un- phy and an increase in serial sarcomere number.
iquely regulates Af and the serial sarcomere number, Since we are dealing with isolated muscle fibers,
fibers were cultured (Jaspers et al., 2004) at positive paracrine factors from neighboring muscle fibers
overall strain (i.e.  5% over ‘ 2.3 mm, referred to as are absent, but not from fibrocytes within the en-
‘‘long fibers’’ below) or negative overall strain (i.e. domysium or micro-tendon.
 20% below ‘ 2.3 mm, referred to as ‘‘short fibers’’ It could be argued that autocrine growth factors
below). The culture period varied from 4 to 97 days. may have been washed away by continuously pump-
Note that long fibers are cultured at sarcomere ing fresh culture medium through the culture cham-
lengths over 2.4 mm, i.e. a length at which Ca21 influx ber. However, if one would suppose that during
from the ECM into the cytoplasm occurs (shown in culture growth factors were washed out, one would
frog fibers). For the long fibers, cultured up to 17 expect long muscle fibers to atrophy, and particularly
days, we did not detect any sign of hypertrophy as short muscle fibers to atrophy and reduce their serial
tetanic force remained stable or decreased slowly. sarcomere number. This clearly did not occur.
Also, the serial sarcomere number showed no major As the basal lamina is expected to facilitate the
effects of high strain. However, after 2 weeks of presence of an unstirred layer near the sarcolemma of
culture for two fibers, which were characterized by the already gel-like ECM, the direct flow of perfusate
lower half-relaxation times than the others, a sig- is expected to pass predominantly on the outside of
nificant, but small, increase in the serial sarcomere the endomysial tunnel of the isolated muscle fiber. In
number of approximately 4% was observed. For addition, generally, the sulphated glycosaminoglycan
short fibers the serial sarcomere number remained branches of the giant proteoglycan molecules (Vlo-
unchanged, even after 97 days of culture. Surpris- davsky et al., 1987; Vukicevic et al., 1992; Mason,
ingly, tetanic force doubled over this period. How- 1994; Tatsumi et al., 1998, as well as collagen IV

371
Huijing & Jaspers
(Vukicevic et al., 1992) within the basal lamina bind This could affect the muscle fiber in two ways. The
several types of growth factors, i.e. the basal lamina removal of high local strains from the complex of
acts as an anchor place for growth factors. Therefore, cytoskeleton, basal lamina and endomysium of the
it also acts as a source of growth factors if concen- muscle fiber would: (1) alter mechanical signalling at
trations fall and hence the basal lamina regulates the the myonuclei according to the paradigm of tensegr-
growth factor concentration. Therefore, a reduction ity (i.e. by changing cell shape or the shape of the
in the concentration of endocrine growth factors, if tensegrity unit of the muscle fiber leading to for
any, in the neighborhood of their sarcolemmal re- example expression of growth factors) and (2) alter-
ceptors will not dramatic. There may be one notable natively, prevent exceeding the threshold for activa-
exception: binding of IGF-1 to heparan sulfate was tion of signalling molecules at the sarcolemma
not shown (Vukicevic et al., 1992). leading to biochemical signalling within the muscle
Therefore, it cannot be excluded that the concen- fiber to the nucleus and eventually enhanced expres-
tration of IGF-1 could have been lowered, because of sion of for instance growth factors.
the absence of other binding proteins, other than When secreted into the ECM, such growth factors,
most common constituents of the ECM (e.g. IGF-1 in turn, will stimulate proteins synthesis over a more
binding proteins; Florini et al., 1996, or serum extended area along the length of the muscle fiber
proteins such as a1-acid glycoprotein and albumin; (autocrine effects) as well as that of its neighboring
Lovich et al., 2001 and specific plasma globulins; fibers (paracrine effects).
Pardridge, 1981). It should be noted that albumin It may be somewhat surprising that, if mechano-
concentration increased within the ECM of mouse signalling within the muscle fiber leads to the expres-
soleus muscle and rabbit tibialis muscle in response sion of growth factors, secretion to the ECM is still
to immobilization (Wagatsuma & Yamada, 2000) needed to activate biochemical-signalling pathways.
and during low-frequency chronic stimulation (Hei- In such reasoning, we hypothesize that the secretion
lig & Pette, 1988), respectively. of growth factors has as a major side effect to
coordinate the adaptation of different units along
the length of the muscle fiber and neighboring muscle
Mechanical factors cells. This would mean that effects of myofascially
induced, variation of local tensile or shear strains
The question that arises is whether isolation of a along the muscle fiber as expected in vivo are aver-
single muscle fiber for the culture does not remove aged so that the degree of adaptation of a substantial
the specific mechanical signals for adaptation of fiber number of presumed units of adaptation is similar,
size. In view of the very substantial effects of epi- even though differences may be sustained over longer
muscular myofascial force transmission, for example, distances.
on the serial distributions of sarcomere length within For example, the differentially increased MAPK
one muscle fiber (as indicated by the finite-element activity within the fibers of rat medial gastrocnemius
modelling), it is hypothesized that the removal of the muscle according to location within the muscle
tensile and shear effects of neighboring muscle fibers (Csukly et al., 2002) may be related to such myofas-
and neighboring muscle also modified or removed cial effects (see Huijing & Baan, 2001a) of remnants
the mechanical signal for adaptation. If this is true, it of the dissected neurovascular tract.
means that the mechanical interactions, via neigh-
boring muscle fibers and their endomysial–perimysial
stromata, may potentially be major determinants of Overall conclusions and discussion
adaptation of muscle size. It should be noted that
such interactions could originate from extra- or In the present work, we reviewed results from experi-
intramuscular sources, as close as other bundles mental work as well as mathematical modelling for
within the same muscle, or as far away as antagonis- very different levels of muscular organization. In
tic muscles. Not only the forces exerted by those addition, we presented some new experimental re-
sources, but particularly also their altered relative sults on epimuscular myofascial force transmission
positions causing shear deformation of the material in X. leavis and on myofascial force transmission
constituting their interfaces could potentially be between antagonistic rat muscles to complete the
important variables. picture presented.
In view of the very substantial effects of epimus- This review demonstrates that, although insight in
cular myofascial force transmission (for example on the mechanisms underlying adaptation of muscle
serial distributions of sarcomere length, as indicated fiber size is growing fast, our knowledge remains
by the finite-element modelling), it is hypothesized rather limited and often controversy reigns, because
that the mechanical isolation of the muscle fiber of a lack of control over the physiological conditions
decreased the signal for adaptation. imposed by the experiments.

372
Adaptation of muscle size and myofascial force transmission
The lack of experimental control is implicit for specific goals attainable at the specific level of orga-
in vivo work on humans, for which rather little nization is necessary (e.g. major mechanisms are not
control on experimental conditions of a target muscle likely to be discovered in an in vivo experiment). We
is feasible (for example, on degree of activation, on also suggest that uncovering knowledge of the me-
values of locally exerted forces and local strains). On chanisms underlying adaptation of muscular size
the other hand, one of the clear advantages of this requires much better control of experimental condi-
type of work is its immediate applicability. tions, as obtained for example through in vitro
A little more control, but as we argue above for experiments on mature muscle fibers. Sophisticated
adaptation studies not a sufficient degree, is possible techniques must be developed in order to study the
within animal experiments involving muscle bellies effects of mechanical signals that are applied to the
surrounded by their natural context of connective muscle fiber at the basal lamina and sarcolemma or
tissues or, more usually, in maximally dissected directly on the myonuclei and how such signals
muscle in situ. It should be noted that in most trigger adaptation of muscle fiber size directly within
adaptation studies using animal experiments (for the nuclei and/or by interactions with biochemical
example immobilization, surgical interventions) ex- factors at the sarcolemma.
perimental conditions are imposed in vivo, while the In spite of having gained knowledge of fundamen-
adaptation effect is assessed for the fully dissected tal mechanisms of adaptation, the work has only
condition of the in situ muscle. Therefore, we argue started, because we need to consider the question
that adaptation effects should also be assessed with how these mechanisms are affected and how their
myofascial effects active. If this is not done, potential effects are modulated by activity at higher levels of
effects of epimuscular myofascial force transmission muscular organization. For example, how do the
will be neglected. A clear example can be derived distributions of sarcomere lengths likely to be present
from observations in patients suffering from spastic within a muscle fiber that is surrounded by adjacent
paresis of the flexor carpi ulnaris muscle (FCU). This muscle fibers of the same muscle or by nearby fibers
affliction leads to the typical palmar flexed and ulnar of other muscles and extramuscular connective tis-
abduction position of the wrist. After dissection of sues affect the processes of adaptation?
the muscle for approximately 50% along the length Therefore, knowledge on fundamental adaptation
of the muscle belly (necessary to allow transfer of the mechanisms should be tested for recognition of effects
FCU tendon insertion to the extensor side subse- at the more complex levels of organization. Potential
quent to the experiment), measurements of length– modification of its effects at these higher levels of
force characteristics yielded no indication for the muscular and connective tissue organization should
reason of this contracture (Smeulders et al., 2004b): be quantified. In that way, integration will become
neither high passive forces, nor unusually high mus- possible if results are obtained at all levels of experi-
cle lengths were encountered at muscle–tendon com- mentation.
plex lengths corresponding to the maximal dorsi- There are other types of important interactions
flexion position possible. This leads to the hypothesis between the studies of the different levels of organi-
that the contracture accompanying the spastic par- zation. For example, once aware of the presence of
esis was caused by myofascial interaction of the FCU distributions of serial sarcomere lengths, researchers
with neighboring muscles or other tissues. working at the level of the isolated muscle fiber may
From this example, it is also clear that we need to try to impose such distributions on their isolated
pay much more attention to potential adaptation of cells. In other words, there should be two-way traffic
the connective tissues within and surrounding the of information and ideas in people studying the
muscle and its acute and long-term effects on mus- different levels of muscular organization.
cular properties. It is clear that non-invasive imaging in human
The discrepancy between in vivo and in vitro data subjects or patients will need to play a very important
on adaptation presented in paragraphs above (e.g. role in the process of recognition of effects at the
effects on muscle fiber diameter and serial sarcomere highest level of organization of tissues. Presently, two
number in whole muscle kept at low lengths, but not types of techniques are available for that: ultrasound
in isolated muscle fibers cultured under such condi- imaging and MRI. The former has the advantage of
tions) demonstrates the value of contrasting ap- being much cheaper and more easily applied than the
proaches at the extremes of the muscular organi- latter. However, the question whether ultrasound
zation (i.e. single fiber in vitro vs whole muscle in its imaging has sufficient contrast and resolution to
natural surroundings). play a major role at the process of recognition of
We argue here that we do need all levels of effects of more fundamental mechanisms at the
experimentation presented, but that a better integra- in vivo level should be raised. MRI also has its
tion of results and developed knowledge is necessary. limitations regarding contrast, but application of
In addition, a better tuning of the experiments to the advanced radio-frequency techniques to give the

373
Huijing & Jaspers
tissue magnetic properties that can be recognized may be quite necessary to recognize the mechanism
within the image may help overcome this problem. of findings– based on animal experimentation.
It is very unlikely that much mechanism will be Finite-element modelling may help to study fea-
uncovered in studies performed at the in vivo level. tures at any level of organization that are inaccessible
Epimuscular myofascial force transmission is a good to direct experimentation and can be used also to
example in this case: in vivo imaging had been around feed and support the theoretical framework within
for years, without leading to any discovery of myo- which results are to be interpreted.
fascial effects, because measurement of locally ex- If successful, the suggested integrative multilevel
erted muscle forces was essential to its recognition approach to the study of muscular adaptation is
and proof. The in vivo shifts of muscles (or their expected to lead also more directly toward applica-
parts) with respect to each other and with respect to tions that will allow manipulation of adaptive
neighboring other tissues can be quantified quite well, processes for the use in clinical interventions, as
even using ultrasound imaging (e.g. Bojsen-Møller et well as training-related goals. Considering the wide
al., 2004). However, the interpretation of effects of range of studies and disciplines needed for such
such relative movements and changes of relative posi- study of adaptation, close collaboration of many
tion remains very hard, unless information regarding different types of laboratories is needed. The depart-
effects of myofascial movement, obtained from better- ments and faculties to which those laboratories
controlled animal experiments, is integrated. We also belong should also train their students to study at
suggest that imaging and analysis of muscles in planes the interdisciplinary level to such a degree that
of view that are quite unusual today (i.e. imaging communication across discipline boundaries is pos-
planes other than only the mid-longitudinal view of sible and the sometimes present abyss separating the
the muscle showing recognizable fascicle patterns) fundamental and applied scientists is bridged.

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