1.1 GENERAL INTRODUCTION
Skeletal muscles exhibit high plasticity to respond to mechanical load for growth
and maintenance. Rodents subjected to hindlimb unloading characteristics of decline
in muscle mass and myofiber cross-sectional area (CSA). Subsequent reloading
triggers a cascade of events involving mild muscle injury, inflammation, regeneration
and growth, resulting in recovery of muscle mass and CSA. This process is regulated
by intracellular signaling cascades that control transcription and protein translation of
phenotype-specific genes.
Calcineurin (CaN), a Ca2+/calmodulin-dependent phosphatase, mediates the
dephosphorylation of nuclear factor of activated T-cell (NFAT) responsible for muscle
differentiation (Delling et al., 2000) and fiber type transformation(Serrano, 2001). In
studies that examined time-dependent changes in signaling pathways involved in
muscle regeneration in unloading-reloading animal model showed that CaN signaling
is mainly involved at the later stages of muscle remodeling in rats (Sugiura et al.,
2005). Therefore, the CaN-dependent pathway, both spatially and temporally, plays an
important role in modulating muscle regrowth. Calcium (Ca2+) signaling is one of the
key stimuli for muscle myogenic commitment, differentiation and myotube fusion
during muscle regrowth (Przybylski, MacBride, & Kirby, 1989). Canonical transient
receptor potential (TRPC) proteins are candidate channel subunits for store operated
Ca2+ entry. These non-selective cation channels are activated by various
physical(Christensen & Corey, 2007) and chemical(Nilius, Owsianik, Voets, & Peters,
2007) stimuli including mechanical loading(Inoue et al., 2009). TPRC1 is the most
abundant isoform of TRPC channels in skeletal muscle(Vandebrouck, Martin,
Colson-Van Schoor, Debaix, & Gailly, 2002) and have been implicated in mediating
Ca2+ entry in skeletal myoblast migration and differentiation(Louis, Zanou, Van
Schoor, & Gailly, 2008). Therefore, to get the role of TRPC1 and CaN-NFAT pathway
response to mechanical loading, there is a need to know the change of TRPC1 and
CaN-NFAT pathway during unloading induced muscle atrophy and reloading induced
muscle regrowth. Afterwards, the role of TRPC1 during muscle regrowth will also be
investigated with the TRPC1 specific siRNA models during reloading.
Previous studies have described other TRPC subunits involvement in NFAT
activation. For instance, TRPC3 channels were reported to be involved in NFAT
activation in skeletal muscle cells during exercise(Rosenberg et al., 2004). TRPC6 has
been demonstrated to be involved in the regulation of Ca-NFAT signaling for cardiac
hypertrophy and remodeling in response to mechanical stress(Kuwahara et al., 2006).
Based on the evidence regarding TRPC1 and CaN characteristics and function, it is
possible that CaN-NFAT pathway is an important determining factor in regulation of
muscle mass and slow myosin heavy chain for reloading induced muscle regrowth
and this process involvement of TRPC1.
In this review chapter, the characteristics of disused muscle atrophy and
treatment are first addressed. This is followed by an introduction of cellular and
molecular regulation of muscle regrowth with emphasis on the calcineurin-NFAT
pathway. Finally, the function of calcium channel TRPC1 in skeletal muscle and other
TRPC subunits involvement in NFAT activation are discussed. This chapter concludes
with the hypothesis and objectives of the study. The experimental investigations are
divided into three chapters (chapter 2 to 4) each with specific methods, results and
discussion. The last two chapters are general discussion (Chapter 5) and the
conclusion (Chapter 6).
1.2 SKELETAL MUSCLE ATROPHY
1.2.1 Etiology of skeletal muscle atrophy
The etiology of skeletal muscle atrophy occurs from diseases of motor nerves or
the muscle tissue itself. It happens in a large range of diseases and conditions such as
disuse, aging, nutritional disorders, nervous system disease, muscular dystrophy and
cancer related cachexia(Bajotto & Shimomura, 2006). Disuse muscle atrophy arises
when muscle contraction is rare, such as in the condition of immobility, long time bed
rest, spaceflight, therefore leading to muscle protein degradation. This usually
happens to patients that have injuries or diseases where they spend long periods of
time in bed to rest and recuperate.
During prolonged disuse conditions, not only a set of morphological changes
such as decreased in muscle mass, a reduction of muscle fiber CSA, a decreased in the
total number of muscle fibers and slow-to-fast twitch fibers transition happened, but
also there are impaired functional changes such as deceased muscle strength,
decreased mobility and function and increased muscle fatigue (Lang et al., 2010). The
detailed morphological and functional changes of skeletal muscle atrophy will be
addressed in the following sections.
1.2.2 Reduced muscle mass
Immobilizition and chronic bed rest results in a progressive loss of muscle mass.
Three studies have shown that after 10 days of not using the leg muscle, there was a 6%
decrease in the lean mass(Brooks et al., 2008; Kortebein, Ferrando, Lombeida, Wolfe,
& Evans, 2007; Kortebein et al., 2008). It was found a decrease of approximately
350g muscle mass in human leg after 28 days of bed rest (Paddon-Jones et al., 2004).
In animal studies, it was demonstrated that a significant decrease of soleus
muscle mass after 7-day, 14-day of hindlimb suspension, not in 3-day unloading
group on rat (Riley et al., 1990). A rapid loss of wet muscle weight in the period of
0-14 day and a subsequent steady decrease in the next 56 days in soleus muscle was
observed during hindlimb unloading of female rates (Thomason & Booth, 1990).
However, an increase in the interstitial fluid volume in muscle has been observed in
muscle atrophy and it maybe a confounding factor for evaluating atrophic changes.
Thus, direct quantification by measuring the muscle fiber cross-sectional area is a
gold standard to assess muscle atrophy.
1.2.3 Reduced muscle CSA
Disuse muscle atrophy is largely attributable to decreases in the size of muscle
fibers, rather than to muscle fiber damage or loss. Fiber CSA decreases dramatically
in response to muscle atrophy. In human study, longer-term of bed rest resulted in a
more reduction of human soleus fiber CSA (Yamashita-Goto et al., 2001). In animal
studies, fusion of muscle fibers was observed in unloading animals, the recovery of
muscle mass was increased quickly while the recovery of CSA per fiber was really
slow (Itai, Kariya, & Hoshino, 2004). The process of completely recovery CSA may
take 5 weeks after unloading on rats (Mitchell, Mills, & Pavlath, 2002). Meanwhile, a
selective atrophy of slow-twitch fibers was found in response to disuse intervention.
Hindlimb suspension for 7 days resulted in a decline in soleus fiber CSA by 33 % and
16 % in type I and type IIb fibers respectively (Stelzer & Widrick, 2003).
1.2.4 Muscle fiber type transition
It is generally accepted that muscle fiber types can be divided into two main
types: slow twitch (Type I) muscle fiber and fast twitch (Type II) muscle fiber. The
slow twitch fibers are more efficient at using oxygen for generating energy for
continuous, extended muscle contractions over a long time. They use oxidative
metabolism to generate ATP and they are slow to fatigue. While the fast twitch fibers
are better at generating short bursts of strength or speed and use both oxidative
metabolism and anaerobic metabolism depending on the particular sub-type. They are
able to fire more rapidly but fatigue more quickly. Fast twitch fiber can be further
categorized into Type IIa, Type IIx and Type IIb fibers.
Under certain conditions, the myosin heavy chain (MHC) isoforms changes in
the direction of either fast-to-slow or slow-to-fast transition. Increased neuromuscular
activity, mechanical loading and hypothyroidism induce fast-to-slow fiber transition,
whereas reduced neuromuscular activity, mechanical unloading and hyperthyroidism
conditions cause transitions in the slow-to-fast direction (Pette & Staron, 2000). In rat
and mouse soleus muscle, hindlimb unloading results in severe atrophy and a
slow-to-fast phenotype transformation as shown by the modification of MHC isoform
distribution (Sabata, 2013). Stevens(2000) found that 14 days of hindlimb unloading
induced slow-to-fast transitions in rat soleus muscle with a significant decline in the
MHC I isoform and increase in MHC IIa or MHC IIx isoforms (Stevens et al., 2000).
1.2.5 Decreased muscle function
The CSA of muscle determines the amount of force it can generate by defining
the number of sarcomeres which can operate in parallel. The decrease of fiber CSA
leads to a decline in force production, leading to a severe impact to daily functional
activities and sports performance. It is obvious that the onset of muscle disuse results
in a significantly greater relative loss of muscle strength when compared with the
actual loss of muscle mass (Deschenes et al., 2002; Farthing, Krentz, & Magnus,
2009). In human study, The maximum tetanic force (Po), maximum activated force
(Fmax) per CSA decreased after 2 and 4 month of bed rest, represented a reduction of
force-generation capacity after long time muscle wasting (Yamashita-Goto, et al.,
2001). In an animal study, hindlimb unloading for 7 days also performed a reduction
in peak Ca2+-activated force and fiber specific force in mice(Stelzer & Widrick,
2003).
The slow-to-fast fiber transition leads to skeletal muscle to be more susceptible
to fatigue. A human study showed an increased Type IIx fiber type and a decreased
Type I fiber after 8-days unloading, suggesting a transition toward a less fatigue
resistant fiber type (Thorlund et al., 2011). In an animal study, 12 days of hindlimb
suspension in mice resulted in a decrease resistance to muscle fatigue (Arbogast et al.,
2007).
1.2.6 Treatment of skeletal muscle atrophy
Countermeasures such as nutritional strategies, exercise or electrical stimulation
have been suggested as to counteract alterations of muscle mass and function due to
disuse. Resistance exercise (RE), also known as strength training, is an important tool
in the treatment of skeletal muscle atrophy as it promotes positive structural
(hypertrophy and phenotypic changes) and functional (strength and power) adaptive
responses (Nicastro, Zanchi, da Luz, & Lancha Jr, 2011). Although RE was confirmed
to be effective for improving muscle mass and strength, the effectiveness cannot be
maintained if exercise stops. Therefore, though exploring the factors involving in the
signaling and molecular pathways in muscle atrophy, some potential therapeutic
treatments should be developed to provide a more beneficial effect in attenuating
muscle atrophy.
1.2.7 Models of muscle atrophy
Skeletal muscle atrophy occurs from general diseases such as cancer, sepsis,
diabetes and extension of inactivity (Hasselgren & Fischer, 1997). Long periods of
bed rest, limb immobilization, spaceflight, hindlimb suspension and unloading the
diaphragm via mechanical ventilation are models to induce disuse skeletal muscle
atrophy (Powers, Kavazis, & McClung, 2007).
Due to the complexities involved in investigating the mechanisms responsible for
disuse muscle atrophy in humans, numerous experimental animal models have
evolved to simulate unloading conditions that lead to disuse muscle atrophy. Different
kind of skeletal muscle atrophy animal models have different research purpose and
advantages. For instance, animal models of limb immobilization (i.e., casting) have
been used to investigate the impact of muscle inactivity on muscle size and function
(Booth, 1982). Rodent animal models using a tail suspension technique to unload the
hindlimb muscles have been used to simulate prolonged spaceflight or bed rest in
humans (Adams, Caiozzo, & Baldwin, 2003; Thomason & Booth, 1990). In this study,
we use a most common procedure of muscle atrophy model, the hindlimb suspension
model to investigate the intercellular molecular mechanism during muscle disuse
atrophy and regrowth.
The first hindlimb suspension rat model was described in 1979 (Morey, 1979).
The rat body was pulled into a plastic mesh with its head-down to move in a 360° arc.
The front paws reached food and water regularly and rear limbs were unloaded
unrestrained. The responses noted in the suspended animals such as negative water
balance, fluid shift, decreasing bone formation rates, muscle atrophy with increased
catabolism and increased metabolic cost represented that this model mimicked results
from animals exposed to real spaceflight. The second published paper about hindlimb
suspension technique was followed at the next year. The model used a back harnesses
that allowed the rats to use its forelimbs to maneuver in a 140° arc(Musacchia,
Deavers, Meininger, & Davis, 1980). Gastrocnemius muscle weight decreased with 7
days of hindlimb suspension and the atrophied condition was readily reversible after
placed in metabolic cages for recovery from hypokinesia. Later on, the concept of a
tail traction model was brought up by Russian scientists (Ilin & Novikov, 1979). This
model appears to be less stressful to animals than back harnesses, as assessed by
adrenal, thymus and corticosterone levels (Halloran, Bikle, Cone, & Morey-Holton,
1988). The tail suspension model was widely used as a model of disuse, for
investigating physiological changes to unloading and the recovery from
unloading(Akhter, Cullen, Pedersen, Kimmel, & Recker, 1998). The hindlimb
suspension model has been extended to mice to study the musculoskeletal responses
to unloading in some studies(Simske et al., 1994; Simske, Guerra, Greenberg, &
Luttges, 1992; Simske, Luttges, & Wachtel, 1989). A number of adjustments such as
use of smaller cages, inclusion of a device to prevent mice from climbing the
unloading apparatus were made to accommodate the smaller body size and differences
in behavior of mice (Morey-Holton & Globus, 2002).
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