GRAPHICAL ABSTRACT

Atrophic myotubes Healthy myotubes

 GRAPHICAL ABSTRACT

Atrophic myotubes Healthy myotubes

Chapter 1 Introduction

Skeletal muscle is the largest organ of the body which contributes 60 %

of the total body weight and is responsible for providing support and helps in maintaining
posture of the body. Along with this, skeletal muscle aids in basic functions like energy
storage, locomotion and whole body metabolism. There are total 640 muscles present in the
body attached to bones through tendons. Characteristically, skeletal muscles have
numerous peripheral nuclei and striations embraced. The fusion of numerous myoblasts are
responsible for the formation of multinucleated myofibers and the striations corresponds to
the contractile elements myosin and actin filaments which occupies almost 80 % of the
whole skeletal muscle. The number of myofibers is determined at the time of birth and
thereafter no or little change occurs in its number (Alnaqeeb et al., 1986, Klein et al.,
2003). However, the size of myofiber is highly susceptible to a decrease or increase in
response to stimuli like exercise (Maughan et al., 1984), injury, disuse (Berg et al., 1991)
and aging (Lexell et al., 1988, Frontera et al., 2000).To fulfill its changing demands
skeletal muscle often switch in between its different fiber types namely type I, type II.
These fibers differ in their myosin heavy chain isoforms which are again of three types:
MHCI, MHCIIa and MHCIIx corresponding to three myosin ATPases I, IIa and IIx
(Brooke et al., 1970). The type I fibers (red fibers) which are commonly known as
oxidative muscle fibers are characterized by a high density of mitochondria, myoglobin
content as well as are highly vascularised (Ingjer et al., 1979, Jansson et al., 1983).
Basically they utilize oxidative phosphorylation of triglycerides as their source of energy,
produces more amount of ATP as compared to type II fibers (MacIntosh et al., 2006). Due
to this fact, they can produce slow contractions for a longer period of time. They are
innervated by slow motor neurons and thus are also named as slow type muscle fibers.
They are known to produce maximum power output and have high isometric tension/cross-
sectional area as compared to type II fibers (Bottinelli et al., 1996).On the other hand, type
IIB muscle fibers employ glycolysis of ATP and creatine phosphate as their source of
energy. Commonly the type IIB muscle fibers are known as fast twitch glycolytic fibers due
to rapid contractions produced by them which make them fatigue easily. These are
innervated by fast motor neurons and are characterized by the presence of low content of
1
Chapter 1 Introduction

myoglobin, mitochondria and a lower supply of blood vessels (Ingjer et al., 1979, Jansson
et al., 1983). The type IIA is known by the name of intermediate fibers or fast twitch
oxidative fibers. These are innervated by fatigue resistant motor neurons and are marked by
the presence of intermediate level of mitochondria, myoglobin and blood supply. As a
result they use both oxidative and glycolytic pathways as their energy source. All these
fibers and the total net content are tightly regulated through a balance between protein
synthesis and protein degradation pathways in skeletal muscles which often lead to
different physiological conditions. In case of increased muscle mass hypertrophy occurs,
which has a positive impact on the body, whereas atrophy proceeds with decrease in muscle
mass and adversely affect the whole body.

Skeletal muscle atrophy is associated with various diseases including cancer,

diabetes, COPD etc, which ultimately lead to decreased rate of survival and further worsens
their response to the treatment (Pennet et al., 2001). Simultaneously, it is responsible for
reduced mobility and quality of life, and increased mortality. Moreover, it precedes with a
decrease in protein content, shrunken fibers, lower strength and in turn a decline in force
production. The severity of atrophy can be explained by the fact that most of the persons
suffer from atrophy at least once during their lifetime. ICU patients suffer from 10%
reduction in their muscle/per day, such that they lose around 50 % of their mass during
their stay in ICU (Alamdari et al., 2015). Myosin heavy chain is the major structural
protein which gets affected by the deleterious effects of atrophy. In addition various other
proteins like actin, tropomyosin, troponins, α-actinin and desmin (Cohen et al., 2012) faces
degradation as a consequence of atrophy which ultimately lead to skeletal muscle with
compromised function and strength.

In addition to these muscle specific proteins, there are some enzymes which
specify the muscle damage. The release of creatine kinase and lactate dehydrogenase in the
serum or media are one of the indications of damaged and injured muscles. Concomitant to
acting as an indicator of damage, creatine kinase also provide significant information about
the functional status of skeletal muscle. In skeletal muscle creatine kinase helps in
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Chapter 1 Introduction

providing energy for contraction through catalyzing the production of creatine phosphate
from creatine and ATP. In absence or decreased contractile proteins condition, the
intracellular level of creatine kinase automatically starts decreasing leaving the muscle with
declined strength and ability to perform contractions (Baird et al., 2012). Whereas LDH
converts lactate to pyruvate with a concomitant conversion of NADH into NAD.
Furthermore, LDH activity also corresponds to muscle fiber composition (Costill et al.,
1976). Basically both of these enzymes specify membranal damage. Simultaneously,
aldolase, myoglobin, troponin, aspartate aminotransferase, and carbonic anhydrase
represent some other markers to indicate muscle damage.
As discussed above, skeletal muscle membrane plays an important role in
protecting muscle atrophy, as any stress implied to membrane can be transmitted from
membrane to cytoskeletal proteins and thus muscles can be protected. However, in case of
damaged membrane, loss of cytoplasmic proteins as well as influx of extracellular ions
occurs which ultimately lead to shrinkage of muscles. Moreover, membranal damage
stimulates activation of calpain through an elevated level of calcium (Abmayr et al., 2004).
The degradation of cytoskeletal proteins also occurs through the activation of proteolytic
systems. There are five proteolytic systems known to regulate skeletal muscle atrophy (Dutt
et al., 2017). The ubiquition proteosomal system represents the most and majorly involved
proteolytic systemin which E3 ligases play the dominant role. Accordingly, E3 ligases
MuRF-1 and Atrogin-1 are found to up regulated in almost every atrophic model. The UPS
comprised of three types of ligases: E1, E2 and E3 which act in a coordinated manner,
Firstly E1 ligase activates the ubiquitin, which is then transferred by E2 ligase to E3ligase.
The E3 ligase ligates the targeted protein to ubiquitin for degradation by proteosome. But
for UPS to work, intact filament need to be released which is processed by calpain and
caspases. Caspases are cysteine endoproeases that degrade the protein following asparte
residue and are synthesized as zymogen which gets activated due to various atrophic
stimulus. Furthermore, caspase-3 as an effector caspase mediates its atrophic effect through
degradation of actin in actinomyosin complexes, both under in vitro as well as in vivo
conditions. This provides the substrate to UPS for further degradation. In addition, skeletal
3
Chapter 1 Introduction

muscle atrophy also proceeds through myonuclear apoptosis mediated by caspase-3. On the
other hand, calpain being a cysteine protease requires calcium for its activation which
ultimately degrades the cytoskeletal proteins (Williams et al., 1999). Along with this, two
other proteolytic systems namely autophagy and cathepsin-L systems also regulate atrophic
processes. LC3B and beclin are the two important mediators of autophagy induced atrophy
whereby they are responsible for the degradation of proteins by autophagosome. In turn,
lysosomes degraded the targeted proteins. Moreover, lysosomal proteases mediate their
effect independent of autophagy. Cathepsin-L has been known to be the most reported
cathepsin which causes atrophy independent of autophagy. It is able to degrade proteins,
transcription factors and even enzymes. Overall, the coordinated action of all these
proteolytic systems is required to attain the state of decreased protein content in skeletal
muscles.
Similarly, a decrease in protein synthesis can be a reason for lower net protein
content in addition to enhanced protein degradation in atrophy. The IGF1-PI3K-Akt-mTOR
pathway represents the major and most important pathway associated with protein
synthesis. The Akt mediates its effects through phosphorylation and hence activation of
mTOR, due to which inhibitory effect on 4E- binding protein 1 (4E-BP1) gets released that,
leads to protein translation. Moreover, the Akt impedes its effect through negative
regulation of FoxO. The FoxO family in skeletal muscles comprise of 3 isoforms; FoxO1,
FoxO3a and FoxO4. Akt phosphorylate the FoxO proteins which lead it away from targeted
proteins in cytoplasm. Whereas in the absence of anabolic stimulus FoxO remains
dephosphorylated and hence translocate into nucleus to activate MuRF-1 and Atrogin-1
(Sandri et al., 2004). Among these isoforms FoxO3 has been implicated to regulate other
proteolytic systems namely autophagy and UPS as well (Scicchitano et al., 2015).
Although an enhanced protein degradation concomitant with decreased protein
synthesis can be seen in different physiological conditions, but common to all an increase
in ROS and inflammatory cytokines was reported which signifies the relation of these two
in skeletal muscle atrophy. Both these converge on intracellular signaling and in turn affect
the proteolytic systems, synthesis of proteins as well as antioxidant defense systems. In
4
Chapter 1 Introduction

turn, regulation of these two processes is detrimental to net protein content in skeletal
muscles.
Inflammation represents an important contributor in the pathology of various
diseases implicated in skeletal muscle dysfunction. A number of these muscle disorders
including muscular myopathies, dystrophy are predominantly associated with prelevance of
inflammatory mediators mainly proinflammatory cytokines. The acute proinflammation is
necessary for imparting protection of cells, whereas long term inflammation is implicated
in various muscle diseases including skeletal muscle atrophy (Domiziana et al., 2015).
TNF-α has been regarded as the principal cytokine which gets elevated during such
conditions. It is known to act through binding of two types of receptors: type I and type II
receptor. Activation of NFκB due to TNF-α binding is known to be the most prominent
effect of TNF-α. Furthermore, inflammation and neurodenegeration are associated with
TNFR-1 whereas TNFR2 contributes towards tissue regeneration and neuroprotection
(Mccoy et al., 2008). In contrast, in some studies the activation of PI3/Akt pathway was
reported in fibroblasts, cardiac and retinal cells due to TNFR2 activation (Kim et al., 1999)
Reports are available which showed that the TNF-α at lower levels promotes satellite cell
proliferation and differentiation (Li et al., 2003; Kurosaka et al., 2013) whereas its higher
level negatively affect the muscle mass and function (Degens et al., 2010). TNF-α
activation affects both the protein degradation and protein synthesis. Furthermore, it was
reported to activate the major transcription factor involved in skeletal muscle atrophy i.e.
NFκB by both canonical and alternative pathway. TNF-α employs indirect mechanism to
induce atrophy in various invivo models. It can either alter the level of various hormones
which affect the muscle mass, thus makes muscle more sensitive towards these factors or
can induce catabolic cytokines and anorexia. Based on this, there are various mechanisms
through which TNF-α can mediate its effect. One of the mechanisms is based on the ability
of TNF-α to inhibit the regenerative potential of satellite cells and hence inhibits the
differentiation (Layne et al., 1999) as well as repair of myotube. TNF-α on the other hand
can induce apoptosis through TNF-α receptor complex and the Fas-associated protein with
death domain in skeletal muscles (Li et al., 1998) but that is not so important from atrophy
5
Chapter 1 Introduction

point of view. However, the catabolic effect of TNF-α represents the most important
mechanism through which TNF-α induces atrophy in almost every atrophic condition.
Similarly in the case of cultured myotubes, TNF-α was observed to decrease the total
protein content specifically muscle specific proteins like myosin heavy chain. The
degradation of such proteins under TNF-α exposure is not associated with altered synthesis
rate, which further confirm the catabolic effect of TNF-α on muscle specific proteins. It
was observed that TNF-α act in a concentration and time dependant manner and this
catabolic effect can further be accelerated through increasing its concentration without any
significant effect on cell death. Within 30min of exposure, TNF-α phosphorylates and
causes the degradation of NFκB inhibitory protein, Iκ-Bα and this effect is rapid, dose
dependant but decreases with the time. Furthermore, the activation of NFκB due to TNF-α
stimulus affects the gene expression and causes long term effect in skeletal muscles which
is responsible for such decrement in skeletal muscle mass and function.
The NFκB is major transcription factor involved in skeletal muscle atrophy. It deals
with the expression of immunomodulatory genes associated with various inflammatory
diseases, cancer and apoptosis. The P65-P50 heterodimers play a significant role in
activation of NFκB in skeletal muscles (Li et al., 2008). Under normal conditions, it exists
in inactivated form associated with inhibitory proteins termed IκB in cytoplasm. Whereas,
on getting stimulus IκB gets phosphorylated on serine 32 and 36, which ultimately releases
it IκB subunit leading to translocation of NFκB in nucleus. After translocation into nucleus,
NFκB binds to specific promoter or enhancer region. As a result, release of
proinflammatory cytokines and up regulation of proteolytic systems under such atrophic
conditions occurs. However, activation of NFκB shows differential effects under different
conditions. In addition to act as a regulator of inflammation, NFκB also regulates the
process of cellular proliferation, tumorogenesis and apoptosis (Barkett et al., 1999, Rayet et
al., 1999). Moreover, on TNF-α exposure activation of NFκB through an up regulation of
ROS level occurs, which further states that ROS is a major contributor of TNF-α induced
atrophy. Although, ROS acts as a second messenger for TNF-α induced effect, but higher
ROS are also able to activate NFκB independent of TNF-α stimulus. Likewise, the addition
6
Chapter 1 Introduction

of exogenous oxidative stress causing agent activates NFκB through ROS, this fact further
implicates the significance of oxidative stress in the pathogenesis of skeletal muscle
atrophy and implies that oxidative stress itself can cause skeletal muscle atrophy.
Oxidative stress has been implicated in a number of diseases associated with skeletal
muscle dysfunction. Moreover, it is majorly involved in a number of inflammatory
diseases, aging, mechanical unloading as well as strenuous exercise. It mediates its effect
through two ways. One of the pathways disturbs the redox signaling that ultimately alter the
gene expression resulting into muscle loss. Whereas other pathway deals with the post
translation modifications of constituently expressed proteins leading to decreased muscle
mass and function. The increased ROS can affect the targeted proteins directly by nitration,
carbonylation, sulfhydryl oxidation, and formation of nitrotyrosine or 4-hydroxy-2-nonenol
adducts or by indirectly altering the redox signaling proteins like kinase etc. Overall, an
increased ROS production results into elevated level of inflammatory cytokines,
chemokines and affects the antioxidant capacity of the target tissues (Moylan et al., 2007).
Skeletal muscles produce various types of ROS, but NO and superoxides are the
primarily generated ROS in skeletal muscles. The superoxide rapidly gets converted into
hydrogen peroxide. Furthermore in the presence of catalytic metals, they give peroxynitrite
and hydroxyl radicals. Associated with this, presence of ROS mainly H2O2 was observed in
most of the atrophy models, which is quite small to diffuse across most of the membranes
and can oxidize various molecules like sulfhydryl, hydroxyl, sulfoxide easily (Allen et al.,
2000). A plethora of studies further reveal the potency of H2O2 to induce atrophy in vivo as
well as in vitro models. H2O2 mediate its effect mainly through elevation of ROS and
alteration of antioxidant defense system (Kaur et al., 2018).
Although skeletal muscle contains multiple sources of ROS, but mitochondria represents
the most important organelle that frequently generates ROS. Interestingly mitochondria
release 100% H2O2 in skeletal muscles of animals undergoing 14 days immobilization (Min
et al., 2011). Evidences suggest that dysfunctional mitochondria activates the AMPK-FoxO
signaling axis that lead to protein degradation and ultimately results in induction of skeletal
muscle atrophy. Furthermore, mitochondria face much deterioration in skeletal muscles due
7
Chapter 1 Introduction

to higher metabolic rate as well as complex functionality of skeletal muscles. In addition,

an imbalanced mitochondrial fission, fusion, respiration and altered morphology have been
often observed during various states of atrophy. Besides this, an overall reduction in
number and functionality of mitochondria can be seen in atrophic skeletal muscles. Due to
which, mitochondrial specific enzymes gets leaked into cytoplasm because of disintegration
of mitochondrial membrane. Succinate dehydrogenase represents one of such enzyme
which in addition to indicate mitochondrial membrane integrity gives information
regarding oxidative capacity of skeletal muscles.
To deal with increased ROS level, skeletal muscle employs its various enzymatic and
non enzymatic antioxidants in a much synchronized manner (Powers et al., 2011). Whereby
enzymes catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD)
provide the first line of defense against increased oxidative stress. The CAT and GPx are
involved in metabolizing H2O2 whereas SOD aids in H2O2 production through catalyzing
the conversion of superoxide radicals into hydrogen peroxide. In addition, GST contributes
towards detoxification of hydrogen peroxide and other hydroperoxides with the help of
reduced glutathione (GSH).Reduced glutathione on the other hand is an important thiol
which maintains the overall redox status of the cell. It can directly or indirectly scavenge
ROS. The level of GSH is maintained by the regulation of GST, GPx and GR. The GST
and GPx utilizes GSH but GR generates GSH from oxidized glutathione.
In addition to all these endogenously produced antioxidants, the dietary antioxidants have
also been observed to protect against skeletal muscle atrophy. In particular, vitamin E,
vitamin C and carotenoids represent the most widely studied antioxidants in terms of
antiatrophic effect. Thus oxidative stress plays a vital role in pathogenesis of skeletal
muscle atrophy, a number of studies support the above notion whereby supplementation
with oxidant scavengers had prevented or delayed the atrophy (Kondo et al., 1999,
Whidden et al., 1999, Arbogast et al., 2007, McClung et al., 2011). However, in most of
the cases the antioxidants mediate their effect through regulation of proteolytic systems
rather employing their antioxidative function. This fact underlines the importance of
regulation of proteolytic systems under such circumstances.
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Chapter 1 Introduction

Oxidative stress may contribute to skeletal muscle atrophy through activation of

proteolytic systems or due to suppression of protein synthesis (Powers et al., 2011). In both
the cases oxidative stress alters the signaling related with protein metabolism. However,
oxidative stress acts in three ways to enhance proteolysis, firstly imbalanced redox status of
cell may regulate transcriptional activators and activates the autophagy and the proteasome
system through increased expression of proteins involved. It may also work through
increased activity of calpain and caspase-3 (McClung et al., 2009, Smuder et al., 2011)
during disuse of muscles or can oxidize the muscle specific proteins so that they can
become susceptible to proteolysis (Smuder et al., 2010). Moreover an effect of oxidative
stress on protein synthesis was also reported in the form of inhibition of mRNA translation
prior to initiation (Hang et al., 2009). For instance, vitamin E supplementation prevented
the atrophy through an inhibition of proteolytic systems rather than mediating its
antioxidative functions (Servais et al., 2007). On the other hand, Indoxyl sulfate mediates
its effects through increased expression of myostatin and atrogin-1 probably due to elevated
level of ROS as well as inflammatory cytokines, and the effect gets diminished on
antioxidant treatment (Enoki et al., 2016). Although most of the studied antioxidants
proved to be beneficial in skeletal muscle atrophy, but still the effect of antioxidants is not
conclusive and requires further study (Powers et al., 2008). It may be due to the fact that
mere regulation of antioxidant defense system does not impart protection against atrophy;
rather simultaneous regulation of both proteolytic and antioxidative systems is required to
attain healthy and functional myotubes (Kaur et al., 2018). Till now, 20 drugs have been
tested on skeletal muscle atrophy, still only one drug i.e. MDA has been FDA
recommended but that too has a number of side effects, making it unsuitable for treatment.
Keeping the necessity and challenges in mind we scrutinized the literature and came up
with one such potential candidate that can be explored in atrophy model for some potential
positive outcomes i.e. Cinnamaldehyde (CNA).
Cinnamaldehyde is one of the bioactive components of cinnamon, most widely used
as a flavoring as well as fragrance agent. Apart from its domestic uses, CNA has gained
much interest due to its profound impact on various diseases. Under stress conditions, it
9
Chapter 1 Introduction

readily prevents lipid peroxidation (Babu et al., 2014) protein oxidation and nitration.
Additionally, CNA exhibit radical scavenging property comparable to standard antioxidants
BHT and ascorbic acid (Babu et al., 2014). It also mediates its antioxidative effect through
regulating the activities of SOD, GST, GPx, GR, and CAT. Furthermore CNA has shown
its effect on GSH and LPO level (Kaur et al., 2018). The regulatory effect of CNA can also
be seen on proteolytic systems in terms of regulation of cytochrome C, caspase-3 and
caspase-9 as well as on LC3II/LC3I ratio (Lv et al., 2017). Overall it is clear that under
various conditions, CNA mediates its effect through diverse molecular mechanisms
including inhibition of NFκB, ROS production, stabilization of mitochondrial membrane
potential. Besides this, it remains unknown whether its dietary supplementation has any
detrimental effect on protein loss in skeletal muscles.

Based on the above literature, the skeletal muscle atrophy was induced by TNF-α as a
model of inflammation induced atrophy. Furthermore, the present study is designed to
determine the effect of Cinnamaldehyde on C2C12 myotubes in terms of regulation of
proteolytic systems, protein synthesis, antioxidative defense system as well as signaling
mechanism involved. Therefore to investigate the effect of CNA on skeletal muscle atrophy
in C2C12 mouse cell line, we will

1. Determine if there is increase in myotube size in CNA+ proinflammatory cytokine-

treated C2C12 cells as compared to control-, CNA- and cytokine treatments only. We
will also determine the level of creatine kinase (a marker for increase in muscle
atrophy) in all four groups of treated cells.
2. Determine if skeletal muscle specific proteins (MyHCf, sarcomeric actin etc.) are
increased in CNA+ cytokine treated C2C12 cells as compared to TNF-α treated and
CNA-treated cells.
3. Determine if cytokines induced signaling pathways (NF-κB, MuRF-1 etc) are regulated
in CNA+ cytokine treated C2C12 cells.
4. Determine if cytokine induced apoptotic pathways are regulated in CNA+ cytokine
treated C2C12 cells.

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