Impact Of Inflammation On Skeletal Muscle

Published Date: 02 Nov 2017

Supervisors: Dr Adam Lightfoot, Mr Gareth Nye and Professor Anne McArdle

Abstract

Skeletal muscle is comprised of spindle shaped fibres made from myoblasts which fuse to form a multinucleate cell known as a myotube (Srinivas et al., 2007). This fusion event occurs hundreds of times to lead to the formation of mature skeletal muscle fibres (Alberts, 2002).

Mild muscle injury due to exercise or critical illnesses such as cancers and inflammatory diseases leads to the release of cytokines from macrophages as well as the injured muscle. A large array of cytokines coordinate the inflammatory response.

The loss of skeletal muscle can be characterized by its severity . Cachexia is the severe loss of muscle mass seen in chronic disease states and critical illness which leads to the systemic increase of cytokines which is associated with loss of muscle mass and an imbalance between protein synthesis degradation and impaired muscle regeneration .

During ageing we see a progressive increase in circulating levels of inflammatory components. The progressive and inevitable loss of muscle mass we observe during ageing is termed Sarcopenia. Sarcopenia is characterized by a decrease in the number of muscle fibres but also a decrease in the size of the muscle fibres as we age .

We hypothesize that IL-6 among other cytokines are involved in a feedback system whereby the cytokine once released from the muscle acts back on the muscle cell initiating further release to cause inflammation and maybe other controversial characteristics such as anti-inflammatory effects, it may mean that IL-6 is constantly stimulated. This hypothesis is supported by our data.

We measured the fibre diameter of the muscle fibres using light microscopy to look for gross muscle atrophy. Quantitative PCR gave us cycle threshold (CT) values used to calculate the change in expression of the cytokines in question upon doses of IL-6 after a time period of 24 hours. We saw no change in the fibre diameter upon treatment with the cytokine IL-6. With the TNF-α, CXCL-1 and MURF-1 expressions we see little change with treatment which may be due to the incubation time or maybe even the concentration of treatment. IL-6 and Atrogin-1 expression does increase upon IL-6 treatment which corresponds to previous research indicating that IL-6 does cause muscle atrophy and damage.

The CCL-5 expression increases at a high dose of IL-6 which indicates the pro inflammatory effect that we expect to see upon IL-6 treatment . At smaller doses of IL-6 we see a decrease in the expression of CCL-5. There is evidence for the decrease at low dosage which suggests the decrease in expression can have anti-inflammatory effects

We conclude that IL-6 does cause muscle atrophy in skeletal muscle as evidenced by elevated expression of the key atrogene Atrogin-1 although it may be in a dose dependant manner and may depend on the incubation period. We can also conclude that we see evidence for IL-6 to be involved in a feedback system, where IL-6 induces the expression of IL-6 itself from the muscle cell. The IL-6 then acts back on the muscle in a self-perpetuating system to persistently stimulate IL-6 release from the muscle cell and cause further muscle atrophy.

Contents

Summary

Introduction

1.1 Introduction

1.2 The sliding filament theory

1.3 Impact of inflammation on skeletal muscle

1.3.1 Mild injury response to inflammation

1.3.2 Severe response to inflammation

1.3.3 Age related response to inflammation

1.4 Muscle as an endocrine organ

1.4.1 IL-6

1.4.2 The IL-6 pathway

1.4.3 TNF-alpha

1.4.4 IL-7

1.4.5 IL-15

1.4.6 RANTES

1.4.7 CXCL-1

1.5Perspective of inflammation towards ageing

Methods

2.1 Sample preparation

2.1.1 Culture of cells

2.1.2 Cell treatment

2.1.3 Cell counting

2.1.4 Cell harvesting

2.2 Rna extraction from c2c12 cells

2.3 qPCR

2.3.1 qPCR array analysis of RNA to determine changes in inflammatory gene expression in control C2C12 myotubes and myotubes following IL-6 treatment.

2.3.1.1 Reverse transcription of RNA

2.3.1.2 qPCR reactions

Results

3.1 Light microscopy-Fibre diameter

3.2 cytokine expression following IL-6 treatment

3.2.1 Atrogin-1 expression following IL-6 treatment

3.2.2 MURF-1 expression following IL-6 treatment

3.2.3 Il-6 expression following IL-6 treatment

3.2.4 TNF-α expression following IL-6 treatment

3.2.5 CXCL-1 expression following IL-6 treatment

3.2.6 CCL-5 expression following IL-6 treatment

Discussion

4.1 Summary of major findings

4.2 General discussion

4.2.1 Measurement of the fibre diameter of pre-treated and post treated C2C12 muscle fibres.

4.2.2 Atrogin-1 expression

4.2.3 MURF-1 expression

4.2.4 IL-6 expression

4.2.5 TNF-α expression

4.2.6 CXCL-1 expression

4.2.7 CCL-5 expression

4.3 Comparison with other research

4.4 Future Perspectives

4.5 Conclusions

Acknowledgements

References

Appendix

7.1 BCA assay dilution table

7.2 BCA assay plate layout example

7.3 Agarose gel plate layout example

7.4 Example table of calculations for cytokine expression graphs

7.5 Example graph produced from table in section 7.4

Summary

Skeletal muscle cells are composed of many myotubes which fuse together to form a multinucleate myoblast. It is hundreds of these fusion events that lead to the formation of a skeletal muscle fibre. Muscle contracts by a process known as the sliding filament theory of contraction which was first observed by Huxley and Hanson in 1954. Skeletal muscle is an endocrine organ; it releases cytokines which are known as myokines due to being released from the muscle fibre.

We used a BCA assay and PCR to run our samples of treated cells which were probed with antibodies to the cytokines we were looking to be expressed from the muscle cell. We took measurements of the fibre diameter pre and post treatment to look for any changes in the fibre diameter that would indicate muscle atrophy upon treatment with IL-6.

Our data showed very little change in the fibre diameter post treatment which indicated no muscle atrophy had taken place. We saw no increase in the cytokines TNF-α, CXCL-1 and MURF-1 which corresponds to the result that we see no change in fibre diameter. However we did see an increase in the expression of CCL-5, IL-6 and Atrogin-1 which are muscle atrophy associated molecules.

We concluded that the increase in expression of atrophy markers was a sign that muscle atrophy would take place but the process takes longer than 24 hours which was the time of measurement for the cytokine expression and fibre diameter measurements. We also said that the lack of increase in certain cytokines was also due to the time period not being long enough and we would extend this to days and possibly even weeks on repeating the experiment.

CHAPTER ONE

INTRODUCTION

1.1 Introduction

Skeletal muscle is a robust and plastic organ which accounts for 50% of the total protein content of the body and is the main driving force responsible for ambulatory movement of the human body . Skeletal muscle fibres can be very large, in adult humans fibres can reach 2-3cm in length and 100 mm in diameter . Skeletal muscle is characterized by its striated appearance due to the thousands of repeating sarcomere units rich in actin and myosin filaments amongst a family of contractile proteins; the sarcomere is the force generating component of skeletal muscle.

Skeletal muscle is made up of spindle shaped muscle fibres based on myoblasts which are the precursor cells that fuse together to form a multinucleate cell known as myotube cells . The myoblast fusion event occurs hundreds of times, which leads to the formation of the mature skeletal muscle fibre (Alberts, 2002).

Differentiating myoblasts act cooperatively by secreting factors which stimulate further myoblast differentiation which signals to neighbouring fibres to encourage differentiation . In healthy individuals muscle fibres are held in position in a meshwork of connective tissue formed by structures known as fibroblasts . This meshwork frame is the supporting control structure for the growth and development of mature muscle fibres. .

When examined using light microscopy cross sections of skeletal muscle fibres appear circular, within this circular outline are many nuclei just beneath the surface membrane known as the sarcolemma . The sarcolemma is the skeletal muscle cell membrane of a muscle cell consisting of a plasma cell membrane and an outer coat made of a thin polysaccharide material that contains thin collagen fibrils, the sarcolemma also plays a crucial role in skeletal muscle structure and function .

1.2 The sliding filament theory

The molecular basis for muscle contraction were observed using elegant studies which showed the interaction of the filaments upon contraction and in a relaxed state

Figure 1: Shows the main components of the contracting muscle such as the myosin(thick) and actin(thin) components alongside the sliding filament components in a relaxed and the changes in appearance of the internal components in a contracted state. (adapted from

The muscle contraction begins with the arrival of a nerve impulse at the neuromusclular junction, which causes the release of acetylcholine. Acetylcholine release causes the depolarisation of the motor end plate which travels through the muscle via the transverse tubules resulting in calcium release from the Sarcoplasmic Reticulum (. The calcium released from the sarcoplasmic reticulum binds to troponin which causes a shape change in the troponin molecule which removes tropomyosin from blocking the active site of the actin molecule, allowing for the Myosin to attach to the actin molecule and form a cross bridge . ATP is then broken down which provides energy for the myosin filaments to pull the actin along its length and shorten the muscle; this occurs along the whole length of the myofibril in the muscle cells. The final step in this cycle is the detachment of the myosin head from the actin filament; this step is sped up by ATP. The ATP molecule is then broken down and the myosin head is re set ready for the next power stroke .

1.3 Impact of inflammation on skeletal muscle

1.3.1 Mild muscle injury-response to inflammation

The inflammatory response is dependent on two factors, firstly the severity of the injury and secondly the degree of muscular vascularisation at the time of the injury . Exercise or mild disease states cause the release of inflammatory cytokines from primary healing and protective cells known as macrophages and fibroblasts as well as the damaged muscle, this release leads on to a small amount of muscle atrophy. The cytokines that are released induce and coordinate the inflammatory response themselves. Cytokines such as TNF-α and IL-6 are released by injured muscle cells such as those in certain disease states and these cytokines can directly induce inflammation . Mast cells are a primary source of cytokines and mast cells can accumulate at the site of injury to initiate an inflammatory response .

1.3.2 Severe Muscular inflammation response

Cachexia is the nutritionally irreversible, loss of muscle which occurs via the loss of cellular fluid into the extracellular space due to severe disease states. This type of muscle loss is seen to develop in a number of chronic pulmonary and non-pulmonary disease states such as chronic obstructive pulmonary disease, cancer , cardiovascular failure and critical illness .

It has been shown in vivo and ex vivo with the soleus, gastrocnemius, and plantaris muscles in human experimental models that with chronic disease we see an underlying increase in the level of inflammatory cytokines and chemokines in the circulation which is associated with loss of muscle mass . Systemic increases in the levels of circulating cytokines are able to induce muscle wasting by the combined effects of creating an imbalance in muscle protein synthesis and degradation pathways in tandem with impaired muscle regeneration . Macrophage expression is increased in many critical illnesses and disease states including obesity. Systemic elevations in cytokine signalling molecules such as JNK proteins and TNF-α mRNA lead to the inflammation we see in damaged muscles which leads to muscle atrophy associated with critical illness and diseases. A rise in the number of inflammatory cytokines can have a deleterious snowballing effect on our health, for example the increase in cytokines due to obesity can then lead to alterations in blood glucose levels and type 2 diabetes which leads to further muscle wasting. .

TNF-α is a cytokine which can mediate the inflammation we see in certain disease states such as obesity. TNF-α has a strong correlation with increases in muscle wasting associated with cancer cachexia. When cancer cachexia is induced in model systems there is an increase in TNF-α along with TNF-α receptors, conclusions from these observations show the increase in TNF-α and more so the increase in TNF-α receptors at the skeletal muscle level are associated with muscle wasting in for example obesity as mentioned in the previous paragraph.

1.3.3 Age related inflammation

Generally in the elderly we see an increase in the levels of circulating cytokines due to the release of cytokines from adipocytes and fibroblasts, leading to an increased inflammatory state in skeletal muscle. Sarcopenia is the degenerative loss of muscle mass and strength with age . In sarcopenia we see a decrease in muscle mass which can be seen as a fall in the number of muscle fibres (type 1 and type 2), the size of the actual muscle fibre decreases and the sarcoplasmic reticulum and T-tubular system which is involved in storing calcium ions and triggering action potentials for muscle contraction proliferate which is related to the loss of contractile elements in the muscle . Many factors contribute to the degeneration of muscle mass with age for example the chronic elevation of cytokines in the circulation which are generally associated with ageing and can lead to increased oxidative stress. The decrease in activity levels and appetite with age will also contribute to the loss of lean muscle mass and strength. Importantly the increase in cytokines with age have been further reviewed and it has been shown that TNF-α, an apoptosis signalling molecule, works more effectively in elderly subjects than in young subjects, this may be due to an increase in cytokine levels (IL-6 and TGF-β) with age which causes a loss of muscle mass and therefore increases the sensitivity of the muscle cell to TNF-α.

1.4 Muscle as an endocrine organ

More recently, the finding that skeletal muscles express myokines suggests that muscle may also be a major source of cytokine secretion. Skeletal muscle is an endocrine organ due to the secretion of hormone-like factors which influence metabolism in surrounding tissues and organs. Myokines are defined as cytokines which are produced and released from muscle fibres and further undergo autocrine, paracrine or endocrine effects on surrounding cells.

Studies have shown that adipose tissue is a major source of cytokine release, releasing adipokines. Myokines play a pivotal role in muscle communication, it is thought that a contracting skeletal muscle will release myokines, which work in a hormone-like fashion.

1.4.1 IL-6

Around a decade ago it became clear that prolonged skeletal muscle contraction initiated the release of IL-6 from muscle fibres into the circulation . The main source of IL-6 in the circulation is the skeletal muscle although studies have revealed skeletal muscle is not the only source of IL-6 in the circulation . Further research has shown IL-6 to be a crucial player in cellular metabolism.

It has been shown that IL-6 is produced by the peritendonous tissue in active muscle during exercise. This work was followed up by an investigation into which muscle cells produce the cytokine IL-6. The study isolated nuclei from muscle biopsies taken before, during and after exercise and used RT-PCR to demonstrate that the nuclear transcription rate for IL-6 increases rapidly during exercise which can be related to illnesses such as obesity which may also put strain on the muscle . This finding suggested a factor associated with contraction and overworking of the muscle increases the rate of IL-6 transcription, most probably from the nuclei of myokines as these were the isolated cells in this case.

Analysis of the human Vastus Lateralis muscle fibres, the largest part of the quadriceps Femoris, using immunohistochemical techniques and in situ hybridization provides further evidence that contracting muscle fibres are a source of IL-6 mRNA .

IL-6 signalling in macrophages seems to be dependent upon the activation of the NF-κβ signalling pathway, which can lead to an inflammatory response, whereas muscle cells can produce IL-6 without activating pro inflammatory pathways due to the intramuscular IL-6 expression being regulated by an array of interacting signalling pathways, such as Ca2+/NF of activated T cells. . Muscle cells are known as the dominant source of IL-6 production, IL-6 is produced in different types of muscle cells such as mature myoblasts, cultured myotubes, satellite cells and murine myofibres. The release from the muscle cell is in response to TNF .

As mentioned previously skeletal muscle cells release IL-6 upon contraction, however IL-6 is also released due to a variety of stimulating factors such as Reactive oxygen species present in the circulation, lipopolysaccharides and presence of inflammatory cytokines .

1.4.2 IL6 pathway

Figure 2 The IL-6 signalling pathway and all its major components as described below. ( adapted from sabiosciences.com)

The IL-6 pathway includes a wider range of cellular and physiological responses including inflammation and cell growth. IL-6 signals through a receptor composed of two subunits, an alpha subunit and a GP130 receptor subunit which is shared with other IL-6 associated cytokines.

IL-6 binds to its receptor initiating cellular events including JAK kinase activation and activation of Ras mediated signalling. Activated JAK kinases phosphorylate and activate STAT transcription factors particularly STAT3 and SH2 domains. Phosphorylated STAT3 forms a dimer and translocates into the nucleus to activate transcription of genes containing STAT3 response elements. STAT3 is essential for gp130 survival. SH2 links the cytokine receptor to the Ras/MAP kinase pathway and is essential form mitogenic activity .

The Ras mediated pathway acts through SHC, GRB2 and SOS upstream and MAP kinases downstream activate transcription factors such as NF-IL-6 that can act through their own response elements in the gene. These factors come together to regulate a variety of complex promoters and enhancers that respond to IL-6 and other signalling factors.

IL-6 activates Pi3K which functions cooperatively to achieve maximal anti-apoptotic effects of the IL-6 molecule against TGF-β due to the phosphorylation of the BCL2 family member BAD. Il-6 transduced anti-apoptotic effects are likely to converge to BCL-2 family members which could act upstream of caspase 3. IL-6 blocks TGF-β induced activation of caspase 3.

Il-6 signalling is terminated by the action of tyrosine phosphotase the proteasome and JAK kinase inhibitors as well as internalisation of cytokine receptors via gp130.

1.4.3 TNF-α

TNF-α is a cytokine whose catabolic activity in skeletal muscle may depend on the presence of other inflammatory cytokines such as IL-1, IL-6 and IFN-γ. TNF-α circulates through the body, responding to certain stimuli such as disease or injury and regulates metabolic tissue activity. A combination of IFN-γ and TNF-α is known to cause a down regulation of the muscular protein MyoD in muscle cells . As well as this down regulation of muscular protein, TNF-α is a potent stimulus contributing to muscle wastage and can inhibit lipoprotein lipase activity resulting in cachexia. TNF-α is a catabolic cytokine which becomes more prevalent with ageing and with its ability to signal cell death it is most definitely a contributing factor in age related muscle atrophy also known as sarcopenia . In contrast to IL-6 there are very few papers which mention the role of muscle-derived TNF-α which suggests to me that it has a number of contrasting effects, i.e. catabolic and anabolic on the surroundings cells.

1.4.4 IL-7

Il-7 stimulates the differentiation of multi-potent haematopoietic stem cells in lymphoid progenitor cells and stimulates the proliferation of all cells in the lymphoid lineage. Il-7 has been detected in media conditioned by primary cultures of human myotubes, differentiated from satellite cells. IL-7 has been seen to be expressed at both protein and mRNA levels. IL-7 is expressed along with the Myosin Heavy chain by myotubes.

1.4.5 IL-15

IL-15 is present at basal levels in the skeletal muscle circulation; however studies have shown that we see an increase in the IL-15 response to both ageing and exercise. Based on the results of this study it is thought that the increases in IL-15 mRNA may be an attempt to counteract muscle loss in skeletal muscle of older subjects. IL-15 regulates T cells and Natural Killer cells. Survival signals that maintain memory T cells in the absence of an antigen are provided by IL-15.

1.4.6 CCL5/RANTES

RANTES also known as CCL-5 is a chemokine that plays a role in recruiting leukocytes to inflammatory sites. CCL-5 also induces the proliferation and activation of certain natural killer cells which cause inflammation and muscle atrophy. RANTES is released by macrophages as a response to stress and is known to affect skeletal muscle cells . Rantes is released from the muscle in response to TNF activation.

1.4.7 CXCL-1

CXCL-1 has similar properties to the cytokine IL-8 in that it acts as a neutrophil chemo attractant, where if CXCL-1 is released from the muscle in response to TNF , neutrophils are recruited to the site of inflammation and promote the release of reactive oxygen species therefore causing muscle atrophy. CXCL-1 expression derived from muscle cells is induced upon exercise; this increased expression of CXCL-1 is coupled with increased muscle mRNA expression of VEGF and CD31 which suggests a role for CXCL-1 in muscle angiogenesis. CXCL-1 as well as having mitogenic properties can also decrease the severity of Multiple Sclerosis and may offer a neuro-protective function.

CXCL-1 is an inflammatory cytokine which is similar to IL-8 in that it is a neutrophil chemo attractant. CXCL-1 acts as a mediator of inflammation where upon expression due to muscle damage the CXCL-1 recruits neutrophils to the site of muscle damage; neutrophils promote inflammation by the release of reactive oxygen species which promote muscle breakdown and atrophy.

1.5 Perspective of inflammation towards ageing

It is not unusual to see two to four fold increases in the levels of cytokines such as IL-6 and TNF-α in the circulation of an ageing individual . The increases in cytokine levels occurs naturally even in the absence of disease states, but due to an increase in the basal level of circulating cytokines and therefore a decrease in the immune function we may see an increase in the incidence of disease with age. It must be stressed that the increased low grade level of cytokines in the elderly is clearly a consequence of age-related disease .

Defective skeletal muscle mitochondria play a vital role in the loss of skeletal muscle mass . Functioning mitochondria are the major producers of reactive oxygen species which damage DNA, proteins and lipids if not rapidly extinguished (Peterson et al., 2012). With increasing age we see an increased mutation rate in mitochondrial DNA which leads to a decrease in the mitochondrial enzymes and a reduced total mitochondrial content.

There is a clear association with both increases in the production of reactive oxygen species and decreases in antioxidant capacity with age . Studies have shown that aberrant ROS regulation plays a role in skeletal muscle deterioration with ageing, the study also confirmed that the degeneration of the muscle does not simply occur due to the increased oxidative damage in tissues but also showed that there is increased generation of ROS by mitochondria isolated from muscle tissue of older animals and humans.

Low grade increases in inflammation we see with ageing may be caused by a larger immediate release of cytokines upon muscle damage and a longer recovery phase, which may again lead to an increase in the basal level of cytokines in the circulation. This hypothesis may suggest that the elderly are capable of implementing an adequate immune response however the inflammatory components are not removed in the normal time frame that is expressed in the young and so the extended presence of cytokines in the circulation of the elderly cause further muscle damage (Peake, 2010), similarly the elderly may have a constantly high inflammatory state and are unable to cope with extra assaults from the cytokines and so we see cell death. This extended recovery phase seen in the elderly may also be the cause of the increase in chronic low grade inflammation we associate with ageing . The stable low level of cytokines in the circulation may be present to enable the body to mount a greater response than normal towards infection to eradicate the damage there and then. The low grade cytokine levels may also be a protective mechanism against the potential risk of further cell damage.

36-48 hours after skeletal muscle injury in mice, it has been demonstrated that there is a significant increase in the release of TNF-α in old mice compared to young mice. It was also found within the same time frame that there is an increase in the levels of IL-6 in the muscle fibres of old mice which was greater than that found in young mice at the same time point. The exaggerated increase in both TNF-α and IL-6 in older subjects upon cell injury shows how the degradation of muscles with age has a profound effect on recovery and ability to cope with muscle injury in the elderly compared with the young (van der Poel, 2011).

As mentioned earlier there is a strong correlation between inflammation and many age related diseases, this correlation has led to the investigation and development of a number of disease related interventions to reduce the inflammation associated with ageing. One alternative pharmacological intervention is the use of medication such as Angiotensin Converting Enzymes and non-steroidal anti-inflammatory drugs among others which play a clinical role in reducing inflammation . The side effects associated with these drugs coupled with the frailty of the ageing immune system, as well as the financial burden have limited the use of these treatments . Therefore changes in lifestyle of the elderly such as exercise training or dietary modifications may be the effective long term alternative to limit inflammation in the elderly. Reductions in calorie intake resulting in weight loss may provide one mechanism to dampen age-related inflammation .. Many, studies have demonstrated that regularly performed cardiovascular exercise training may reduce markers of systemic inflammation .

We see with increases in age a common theme of increases in circulating cytokine levels, which ultimately leads to inflammation and muscle wasting. The increases in cytokines with age have an effect on the mitochondria and cause mitochondria to become defective and therefore produce ROS which have a deleterious effect on the muscle. The increase in circulating cytokines is coupled with a worsening immune system, together these two factors contribute to muscle wasting that we commonly see with age.

Chapter Two

Methods

sample preparation

Culture of cells

Dulbeccos’s Modified Eagles Medium DMEM (Sigma Aldrich, Dorset, UK).

Fetal Calf serum (FCS) (Sigma Aldrich, Dorset, UK).

L-Glutamine (Lonza, UK).

Streptomycin (Sigma Aldrich, Dorset, UK).

Horse serum (Lonza, UK).

Phosphate Buffered Saline PBS (Sigma Aldrich, Dorset, UK).

C2C12 cells were grown in cell culture medium comprising DMEM, supplemented with 10% Fetal Calf serum (FCS), 5ml of 2Mm L-glutamine and 5ml 50 μg/ml streptomycin. Cells were grown in a controlled environment of 37 oC and at 5 % CO2 rich humidified environment. Initial cell culture was carried out in T75 (Costar, London, UK) tissue culture flasks and further sub-cultured into 6 well plates (Costar, London, UK) for treatment. Myotube formation in the C2C12 cell line was induced by replacing the 10% FCS containing media with DMEM containing 2% Horse Serum(HS) with 5ml of 2Mm L-Glutamine over a five day period. This five day treatment period with 2% HS encouraged Skeletal muscle myoblast differentiation to form myotubes . Differentiation was assessed periodically throughout the 5 day period by light microscopy.

Cell Treatment

IL-6

C2C12 cells were treated with different doses of IL-6, CCL-5, CXCL-1 and CCL-2. The different concentrations contained 2% Horse serum and 5ml Glutamine and the cytokine in question to make up a control with 0 μl and 5, 10 and 20 ng/ml concentrations of treatment. Cells were incubated at 37°C for 24 hours. Cells were harvested and the cell pellet was isolated and stored for analysis by western blotting, qPCR, ELISA and we will use separate cell cultures for LIVE/DEAD analysis.

2.1.3 Cell counting

0.4% trypan blue solution (Sigma Aldrich, Dorset, UK).

Phosphate Buffered Saline PBS (Sigma Aldrich, Dorset, UK).

Suspend the cells in 1 ml of trypsinized cells in 9 ml of complete media. The cell suspension is transferred into a 1.5ml microfuge tube before adding300 μl of PBS and 500 μl of 0.4% Trypan blue solution to the cell suspension. Mix thoroughly and allow to stand for 5 to 15 minutes.

With a cover-slip in place, transfer a small amount of the trypan blue-cell suspension to a chamber on the hemocytometer (shown in figure 3).

Figure 4 shows a haemocytometer grid as seen under the light microscope.

Count all the cells (non-viable cells stain blue, viable cells will remain opaque) in the 1mm centre square and the four corner squares. Keep a separate count of viable and non-viable cells. If greater than 25% of cells are non-viable, the culture is not being maintained on the appropriate amount of media; reincubate culture and adjust the volume of media according to the confluency of the cells and the appearance of the media. If there are less than 50 or more than 200 cells per large square, repeat the procedure adjusting to an appropriate dilution factor. Repeat the count using the other chamber of the hemacytometer. Each square of the hemacytometer (with cover slip in place) represents a total volume of 0.1 mm3 or 10-4 cm3. Since 1 cm3 is equivalent to 1 ml, the subsequent cell concentration per ml (and the total number of cells) can be determined

2.1.4 Cell Harvesting

Phosphate Buffered Saline PBS (Sigma Aldrich, Dorset, UK).

The growth media containing 2% Horse Serum(HS) with 5ml of 2Mm L-Glutamine was removed before 1ml of PBS was added to each well. C2C12 myotubes were then disturbed and scraped from the bottom of the 6-well tissue culture plates (Costar, UK) using a cell scraper. The myotubes now suspended in the PBS were then removed, 500μl into two separate eppendorfs for each well. One eppendorf can be used for RNA analysis and one for protein analysis.

2.2 Extraction of RNA from C2C12 cells

• TriReagent (Sigma Aldrich, Dorset, UK).

• Isopropanol (Sigma Aldrich, Dorset, UK).

• Chloroform (Sigma Aldrich, Dorset, UK).

• Ethanol (75%)

• Dulbecco’s Phosphate buffered saline (DPBS) (Sigma Aldrich, Dorset, UK).

C2C12 myotubes were cultured in vitro as described in Section 1.1. C2C12 myotubes were harvested using a cell scraper. The cell pellet was re-suspended into TriReagent and allowed to stand at room temperature for 5mins. Two hundred microlitres of chloroform was added to each sample and agitated at room temperature for 15 seconds then allowed to stand for a further 15mins. Following incubation samples were centrifuged at 12,000g for 15mins at 4oC to separate the lysate into several phases: The cell lysate was separated into 3 phases, a lower (pink) organic protein containing phase, a DNA containing interphase and an upper (colourless) RNA containing aqueous phase.

The upper RNA phase was aspirated into a new tube from each sample and combined with 500μl of isopropanol. Samples were vortexed and incubated at room temperature for 10mins then centrifuged at 12,000g for 10mins at 4oC. The supernatant was aspirated and discarded for each sample and the RNA pellet washed with 75% ethanol and centrifuged at 12,000g for 5mins at 4oC. Supernatants were aspirated and discarded, and pellets air dried at room temperature for 5mins. Finally, the RNA pellet was re-suspended into DNase/RNase free ddH2O, re-suspension was obtained by repeated pipetting and a brief heating step to 55oC for 15mins. Samples were then stored at -80°C.

2.3 qPCR

Quantitative PCR is limited as it only allows measurement of a PCR end product, however this measurement is limited upon the quantity of reagents present in the reaction as the quantification is made during the "lag" phase of amplification. In qPCR the product is measured during the exponential phase of amplification, and thus the amount of starting cDNA calculated.

The quantification step of qPCR is reliant on a chemical reaction during the amplification cycles using the SYBR Green I fluorescent chemistry. The template cDNA is copied and molecules of the fluorescent chemical SYBR Green I are incorporated into the minor groove of the double-stranded DNA. The incorporation of the SYBR Green I into the DNA strands is typified by an increase in fluorescence, further amplicon copies produced by each PCR cycle results in amplification of the fluorescence intensity. The fluorescence intensity is analysed at the end of each PCR cycle, and is represented graphically, plotting the fluorescence intensity against the cycle number. By doing this, the starting quantity of cDNA can be calculated from the cycle at which the fluorescence intensity increases above a pre-determined "threshold" known as the cycle threshold.

Once the PCR reaction is completed a melt or dissociation curve (Figure 5) can be produced. This analysis allows determination of the specificity of the primer annealing and amplification stages of the PCR reaction. The denaturation of the double stranded DNA occurs at varying temperatures based upon the strand length and its guanine/cytosine composition. It is possible to analyse several different amplicon products produced in one reaction. The several products may be due to splice variants in the gene being analysed, unspecific primer annealing and primer dimerisation. However, quantification and analysis can only occur if only one product is amplified which is shown by a single peak.

Figure 5 Example of a qPCR melt curve showing amplification of individual genes of interest, giving an indication of primer affinity and the overall denaturing and annealing process.

2.3.1 qPCR array analysis of RNA to determine changes in inflammatory gene expression in control C2C12 myotubes and myotubes following IL-6 treatment.

Quantitative Polymerase Chain Reaction arrays (qPCR) were used to analyse changes in inflammatory gene expression in C2C12 myotubes.

2.3.1.1 Reverse transcription of RNA

RT2 first strand synthesis kit (SABiosciences, Maryland, USA).

5X Genomic Elimination Buffer (SABiosciences, Maryland, USA).

RNase-free H2O (Invitrogen, Paisley, UK).

Isolated RNA was reverse transcribed to cDNA to allow analysis in qPCR reactions. The transcription process was carried out using RT2 first strand kit. Firstly contaminating genomic DNA was removed by combination of 2mg of template RNA with 5x genomic DNA elimination buffer with RNase free H2O up to a total volume of 10l. To ensure successful depletion of gDNA samples were analysed using a spectrophotometer at 260/280nm for the presence of gDNA (Eppendorf, Biophotometer, USA). The reaction mixture was prepared and heated to 42oC for 5mins and immediately cooled on ice for 1 minute. A reverse transcription reaction mixture was prepared as follows for each 10l gDNA elimination mixture:

4l 5X RT Buffer 3

1l Primer & External Control Mix

2l RT Enzyme mix 3

3l RNase-free H2O

Using a PCR thermocycler (Bio-Rad, iCycler, Bio-Rad, Hercules) the reaction mixture was then heated to 42oC for 15mins, following which, heating to 95oC for 5mins degraded the RNA and activated the reverse transcriptase enzyme. The reaction mixtures were then stored at -20oC.

2.3.1.2 qPCR reactions

Superarrays RT2 qPCR Master Mix

RNase-free dH2O

cDNA

The PCR reaction was carried out using a Bio-Rad thermocycler. cDNA was combined with the following mixture:

20l of 0.5g cDNA

1305l RNase-free dH2O

1375l of RT2 qPCR Master Mix (SABiosciences, USA).

Twenty five microlitres of reaction mixture was added to each well of the 96-wells of the array plate. The plate was then securely sealed and subject to the following experimental cycles in a thermocycler (Figure 2.6):

1 cycle for 10mins at 95oC

40 cycles. Each cycle comprises 15 seconds at 95oC followed by 30 to 40 sec at 55oC and 30 sec at 72oC

The fluorescence from the SYBR green incorporation was quantified throughout each cycle to obtain the threshold cycle (Ct) for each gene. Quantification of changes in gene expression was carried out using the delta-delta ct (ΔΔct) method. This method calculates changes in gene expression between the control and experimental samples in both the house keeping genes and genes of interest, expressing data as fold change.

Atrogin-1

TNF-α

62, 61.6,60.6,59.2, 57.1,55.7, 54.7, 54

62, 61.6,60.6, 59.2,57.1,55.7, 54.7,54

MuRF-1

62, 61.6, 60.6, 59.2, 57.1,55.7, 54.7, 54

Figure 6 is images of Agarose gels which show temperature gradients for the optimal annealing temperatures of the primers in question. The annealing temperatures are in degrees Celsius at the base of each of the images. The temperature gradient for TNF-α, MURF-1 and Atrogin-1 are shown.

Chapter Three

Results

Figure 3.1 Light microscopy Fibre diameter

A

B Penner + Muder - Presence Of Another Man (Round Table Knights Remix)

A

Penner + Muder - Presence Of Another Man (Round Table Knights Remix)

C

Penner + Muder - Presence Of Another Man (Round Table Knights Remix)

C

Figure 3.1 C2C12 myotubes viewed using light microscopy before being treated with IL-6 (A) and post treatment with IL-6(20ng/ml)(B). (C) Graphical represntaion depicting the average myotube diameter for control untreated and myotube treated with IL-6 for 24 hours after 24 hours. Data are a % of the control +/- SEM (n=10).

There is no significant change in fibre diameter pre-treatment (A) or post treatment (B) which is shown in figure (C).

3.2 Cytokine expression upon IL-6 treatment

Figure 3.2.1 Atrogin-1 expression from C2C12 muscle myotubes following IL-6 treatment. Data shown as 2-AΔCT values +/- SEM, P ≤ 0.05 (n=4)

Following treatment with IL-6(5, 10, 20 ng/ml) for 24 hours, Atrogin-1 expression increases in a dose dependant manner. Following 5ng/ml treatment we see a fivefold increase, after 10ng/ml treatment we see a tenfold increase and following 20 ng/ml treatments there is an 18 fold increase in Atrogin-1 expression.

Figure 3.2.2 MURF-1 expression from C2C12 myotubes following IL-6 treatment.

Data shown as 2-AΔCT values +/- SEM, P ≤ 0.05 (n=4).

An increasing dose of IL-6 in this case shows no increase in MURF-1 expression. From control to first dose of IL-6 there is no change in MURF-1 expression, however between 5 and 10ng/ml doses we see a decrease in the expression of MURF-1. There is no change in MURF-1 expression between doses 10ng/ml and 20ng/ml.

Figure 3.2.3 Il-6 expression from C2C12 myotubes following IL-6 treatment.

Data shown as 2-AΔCT values +/- SEM, P ≤ 0.05 (n=4).

IL-6 expression upon treatment show a 3 fold increase following a 5ng/ml dose, a 28 fold increase following 10ng/ml treatment and a 50 fold increase following a 20ng/ml dose. We see a large increase in IL-6 expression following IL-6 treatment which correlates with the findings from Atrogin-1 where we also see 10 fold increases in expression following treatment.

Figure 3.2.4 TNF-α expression from C2C12 myotubes following IL-6 treatment.

Data shown as 2-AΔCT values +/- SEM, P ≤ 0.05 (n=4).

The expression of TNF-α was not changed by any dose of IL-6. TNF-α expression correlates with MURF-1 expression following treatment where there is no change in expression with increasing doses of IL-6.

Figure 3.2.5 CXCL-1 expression from C2C12 myotubes following IL-6 treatment.

Data shown as 2-AΔCT values +/- SEM, P ≤ 0.05 (n=4).

CXCL-1 expression does not change with increasing doses of IL-6. This result is supported by the lack of change in TNF- α expression and MURF-1 expression upon IL-6 treatment.

Figure3.2.6 CCL-5 expression from C2C12 myotubes following IL-6 treatment.

Data shown as 2-AΔCT values +/- SEM, P ≤ 0.05 (n=4).

CCL-5 does not increase with small doses of IL-6, but following treatment with 10ng/ml we see a 1 fold decrease in CCL-5 expression and following treatment with 20ng/ml of IL-6 we see a 2 fold increase in CCL-5 expression.

Chapter four

Discussion

4.1 Summary of major findings

Following IL-6 treatment there was no change in myotube diameter over a 24 hour period.

We see an increase in the expression of Atrogin-1 following IL-6 treatment in the C2C12 myotubes.

There is no statistical change in MURF-1 expression from C2C12 myotubes following treatment with IL-6.

Treatment of C2C12 myotubes with IL-6 causes a dose dependant increase in IL-6 gene expression.

TNF-α expression from C2C12 myotubes doesn’t change with increasing doses of IL-6.

There is no statistical change in CXCL-1 expression from c2c12 myotubes following treatment with IL-6

CCL-5 expression decreases following treatment with 10ng/ml doses of IL-6 and increases with larger doses of 20ng/ml of IL-6.

4.2 General Discussion

The aim of this project was to look at the impact of inflammation on C2C12 skeletal muscle cells focusing on the cytokine IL-6. We focused mainly on IL-6 treating C2C12 myotubes with varying doses of IL-6. IL-6 is a well-known cytokine released in response to stress and it has deleterious effects on the muscle fibres such as decreases in muscle fibre size and number . We aim to investigate different cytokine effects and pathways including IL-6 to enable the more efficient production of drugs to prevent the action of IL-6 on the muscle cells to help treat diseases and slow ageing of muscle cells.

4.2.1 Measurement of the fibre diameter of pre-treated and post treated C2C12 muscle fibres.

Figure 3.1 shows there are no clear differences in the muscle fibre diameter. The results shown from the fibre diameter measurements go against our hypothesis. Previous studies have shown that IL-6 expression causes atrophy of skeletal muscle fibres and a decrease in the muscle fibre diameter .However the measurements we have taken of fibre diameter post and pre-treatment show no significant difference. After treatment the C2C12 cells were left for 24 hours to allow for the IL-6 to act upon the muscle cell and exert its effects on the fibre, the fact that we did not see a decrease in fibre diameter may show that the 24 hour period may not be a wide enough time frame to observe appreciable changes. Studies performed which have shown a decrease in the muscle fibre diameter have been carried out with a lower IL-6 dosage over a period of two weeks which supports the thought that leaving the treated myotubes for a longer period of time may give us a positive result.. We could repeat the experiment and leave the IL-6 treatment for 48 hours to see if this causes a decrease in the fibre diameter. If there is still no decrease in the fibre diameter after 48 hours then we could increase the dosage of IL-6 to what effect this has on the fibre diameter.

The model we used is a powerful but acute treatment more akin to a critical illness situation. Whereas a more longitudinal study is more akin to the progressive inflammation during ageing. Based on these findings our data suggest that the severe acute inflammation in the critically ill, accompanied by profound muscle wasting is not directly mediated by IL-6. These findings support previous work which demonstrates that TNF-α is the main driver in this pathology.

Although the use of cells for this project is suitable and an effective method of research, other model systems may be better suited to a possible long scale, low grade inflammation experiment, a kin to what is seen in the elderly. The use of mice here would closely mimic the conditions seen in the elderly and would therefore allow us to better determine the full effect of IL-6 in muscle wasting.

4.2.2 Atrogin-1 expression

Atrophy in skeletal muscle results from enhanced protein breakdown and is associated with the induction of the muscle specific ubiquitin protein ligases, Atrogin-1.

Atrogin-1 is dramatically expressed in skeletal muscle atrophy linked to critical illness and endotoxicicty so it is no surprise that as we increase the IL-6 concentration in a dose dependant manner our results showed a correlating increase in the expression of Atrogin-1.

The increase in the Atrogin-1 expression shown in Figure 3.2.1 does not support the findings from the muscle diameter measurements shown in Figure 3.1. As mentioned previously IL-6 is released from the muscle cell and is known to cause atrophy of muscle fibres, which leads to the expression of Atrogin-1. We see from our data the expression of Atrogin-1 in response to increasing IL-6 doses increases but we do not see any change in the diameter of the muscle fibres and therefore no visual evidence of muscle atrophy. Muscle wasting occurs over an extended period of time due to the breakdown and removal of whole muscle cells at a time. The expression of the atrophy markers such as Atrogin-1 is almost instantaneous following treatmenttherefore the muscle breakdown may have begun but we will need a longer time period to see a noticeable difference in the fibre diameter which may be why we don’t see any change in fibre diameter but do see a change in the expression of Atrogin-1. We see this in previous studies which do show a decrease in fibre diameter where the cells have been left following treatment for weeks rather than hours . Our findings perhaps intimate that IL-6 may play a role in the progressive loss of muscle mass during ageing. The expression of Atrogin-1 occurs very quickly following treatment which suggests the high sensitivity of the Atrogin-1 gene to IL-6, however the rate at which Atrogin-1 initiates muscle wasting is obviously limiting as we see no changes in the fibre diameter after the 24 hour period.

4.2.3 MuRF-1 expression

Atrophy in the skeletal muscle associated with IL-6 increases, contributes to the induction of the muscle ring finger protein or MuRF-1 which is a ubiquitin ligase. Ligases are known to bind and mark muscular proteins for degradation by Ub-dependant proteasome degradation during muscle atrophy .

From our results we do not see an increase in the expression of MuRF-1 upon treatment with IL-6. Previous studies have shown that MuRF-1 is associated with muscle wasting.. If we left our cells following treatment with IL-6 for a longer period of time we may see an increase in MuRF-1 expression. The lack of expression of MuRF-1 correlates with the lack of change in muscle fibre diameter we see in Figure 3.1. This correlation may support the thought of leaving the cells for a longer incubation period, if left for longer we may in fact see a decrease in the fibre diameter along with an increase in MuRF-1 expression which is what we expect. The method of treatment is carried out in a closed system which may prevent interactions between different atrogenes such as MuRF-1 and TNF-α, which interact possibly with catalytic effects .

4.2.4 IL-6 expression

Upon treatment of the cells with IL-6 we see a large increase in the expression of IL-6 from the myotubes. IL-6 is secreted in response to treatment with TNF-α which leads to the degeneration of muscle fibres.

Exogenous IL-6 can fuel many catabolic processes such as reducing pro-inflammatory cytokine up-regulation and neutrophil infiltration.

The IL-6 release induced by IL-6 could be evidence for the role of a self-perpetuating cell as described in figure 2 in chapter one where we see that the release of cytokines such as IL-6 due to muscle damage, act back on the muscle fibre causing the further release of cytokines leading to increased damage to the muscle fibres and possible leakage of the cytokines to effect other pathways such as the autoimmune disease pathway. This feedback system suggests that IL-6 is persistently cycling which may explain the large increases in IL-6 with time and with further extended time I feel muscle atrophy would take place and persistent signalling of IL-6 would have an increasingly detrimental effect on the muscle cell itself.

4.2.5 TNF-α expression

TNF-α expression does not appear to increase in response to IL-6 treatment in culture however it has been shown TNF-α expression increases in response to IL-6 in the body.

TNF- α expression can fuel catabolic processes and limit the effect of pro inflammatory cytokines and therefore reduce muscle wasting.. We could use an ELISA to test for the protein levels of TNF-α as this will enable us to compare the changes or in our case lack of changes in gene expression with protein content.

These differing results suggest that there may be other factors within the body which play a role in the expression of TNF-α. In culture surrounding tissues and possible leakages are removed so the system is stable and free from contamination. We see no correlation between the TNF-α data and IL-6 data, however in the body there is an array of factors which may contribute to the release of TNF-α rather than IL-6. Factors such as critical illness and endotoxemia all cause increases in TNF-α expression. In culture we do not see an increase in TNF-alpha may be a finding which supports the notion that it is these other factors such as increased blood glucose concentration which lead to TNF-alpha muscle wasting, not simply IL-6 expression. With the controlled conditions we see in cell culture it is not surprising we see no change in the release of TNF-α, many papers have suggested the contribution to the presence of TNF is down to surrounding factors such as TNF being released from macrophages.

Figure 7 A schematic showing the pathways of IL-6 and TNF-α are separate and will not have any effect on each other until further downstream. This lack of interaction between the two pathways is supported by the data obtained from our work. Figure adapted from

Cell culture is a closed system so enables us to see the effects of individual cytokines on a specific muscle cell, however our results from cell culture show differences from those that we see in the body, which may not support future research in cell culture as cell culture does not have surrounding factors that are associated with the bodies system and may mean there is less association than we think between cell culture and the way the human body reacts to certain stimuli.

TNF-α expression upon treatment with 5ng/ml shows a large error bar which may indicate poor technique, however the large error is only in one of the treatment groups which suggests variation in this specific time frame. There may be pipetting errors and concentration errors among others which indicate to me that with this TNF-α expression analysis would need repeats to ensure accuracy.

4.2.6 CXCL-1

Following treatment with IL-6 we do not see any change in the expression of CXCL-1 no matter how strong the dose. CXCL-1 is released from Macrophage cells when triggered by a stress response. CXCL-1 signals through its receptor known as the CXCR-2 receptor, this takes an extended period of time than if the molecule were signalling through itself , which leads me to believe if the cells were given a longer time period before analysis we would see a change in the expression of CXCL-1 as the signalling would have occurred through the receptor. This delay in the expression of CXCL-1 may contribute to the fact that we saw no change in the fibre diameter in a time frame of 24 hours.

Previous studies have shown that increases in the levels of CXCL-1 occur following increases in IL-6.. The expression is seen mainly from the liver which is surrounded by blood and cells which may influence the CXCL-1 expression to a greater extent than IL-6 alone in the skeletal muscle. Other related studies show an increase in the expression of CXCL-1 with exercise and relate this with a correlating increase in IL-6. Our mice were not subjected to periods of exercise, simply our cells treated with IL-6 so maybe it is a factor more associated with exercise rather than simply IL-6 which leads to the expression of CXCL-1.

4.2.7 CCL-5

CCL-5 expression does not change with a small dose of 5ng/ml and time period of 24 hours but on increasing the dose to 10ng/ml we see a 1 fold fall in the amount of CCL-5 expressed, this may be due to experimental error as our measurements are temporal and so at this specific time point there may have been a distraction or change in room conditions. It has been shown before that upon small doses CCL-5 can down regulate IL-6 expression.

Following an increased dose of IL-6 we see an increase in the expression of CCCL-5 which is supported by previous studies . The requirement for large doses of IL-6 to cause an increase in the expression of CCL-5 may be an indication of the reduced sensitivity to cytokines as CCL-5 is involved in important processes such as inducing the proliferation of Natural Killer cells which needs to be specific and only activated at specific times.

The observation that we see an increase in CCL-5 expression but no change in fibre diameter may be due to CCL-5 being a recruitment molecule for leukocytes into inflammatory sites. The recruitment process can take time which supports the idea for a longer incubation time and I think this would lead to muscle atrophy and a decrease in fibre diameter in a mouse or human model, however in culture there is nothing to recruit as we have isolated our C2C12 mouse cells in a clean environment so in our case we may not see a change in fibre diameter.

4.3 Comparisons with other research

There are many influential papers which look at the impact of inflammation on skeletal muscle many of which many focus on the impact of the cytokine IL-6 on the muscle cells. There is a strong agreement for the pro inflammatory effect that IL-6 has on the muscle cells, however certain papers have concluded that IL-6 can have anti-inflammatory effects on the muscle cells. This may be due to the variation in techniques and protocols used in the experiments and I mention again as in the previous section that the muscle cells are in culture rather than in the body which according to my results can affect the skeletal muscle response to IL-6 in different ways. Or it may be due to the fact that IL-6 does have a pro inflammatory as well as an anti-inflammatory effect in different conditions but we are not sure which conditions lead IL-6 to have which effect.

The C2C12 mouse cell line is a common muscle cell line used for investigations. Most studies use this cell line which enables good comparisons to be made between studies which tend to give similar results and show that IL-6 is a pro inflammatory cytokine. It is when different cell lines are used or cells are not seen to differentiate in the correct manner where we see indifferent results. For example a previous study which examined the effects of IL-6 on skeletal muscle saw no change in atrogenes whereas our data did show an increase in Atrogin-1 following IL-6 treatment. The differences between the study mentioned and our study were; smaller doses of IL-6 , a longer incubation period and instead of using the C2C12 mouse cells they used female mice known as Sprague-Dawley rats, so although the experiment and comparisons appear the same at first glance there are many differences between the two experiments and hence different results.

Despite the number of papers published, I have seen very few papers which mention the IL-6 pathway in the inflammatory response with regards to there being a feedback system. As IL-6 is released from the skeletal muscle cell in an inflammatory response, it seemingly acts back on the cell it has been released from and causes further release of IL-6 and an increase in the inflammatory response. I think this feedback of IL-6 occurs as from my data we can see IL-6 causes IL-6 release from the muscle cell and upon release from the muscle cell the gp130n cell surface receptor on the muscle attracts the IL-6 released back to the muscle cell surface receptor to cycle back into the cell causing a persistent stimulation of the IL-6 response and therefore constant muscle atrophy.

Figure 8: Skeletal muscle is a secretory organ. The secretory cytokines promote muscle hypertrophy possibly by the feedback system shown above. TNF-α induces inflammation in the skeletal muscle which causes the release of cytokines. It is thought the cytokines then act back on the muscle causing the release of further cytokines causing Muscle atrophy which is detected by the presence of atrophy markers MURF-1 and Atrogin-1.

The results obtained in general show IL-6 is a pro inflammatory cytokine we can see this due to the increased expression of atrophy markers and cytokines upon treatment with IL-6. However some of the responses such as that of MURF-1 and TNF-α, along with the fibre diameter measurements showed an almost anti-inflammatory response to IL-6. IL-6 infusion has been shown previously to inhibit the TNF-α levels thus in turn keeping MuRF expression down due to the lack of TNF-α which is the reason for no change in gene expression.

The contrasting results may show that the protocol being used is inadequate as we see temporal results due to the time frames used. As shown in previous studies if we can extend the time frame we may observer more clear results. This will give us a clear indication of whether the inhibiting factor for the experiment is simply time or is a more elusive factor such as an error in the cell growth stage or an error in the protocol or possibly human error.

4.4 Future perspectives

The research into the impact of IL-6 on skeletal muscle cells is primarily to learn about the IL-6 cytokine in order to get a better understanding of how it causes inflammation in skeletal muscle cells so we can learn how to block the effects of IL-6 in a pharmacological approach. Interventions directed against IL-6 represent excellent targets for cancer and inflammatory diseases, currently there are very few anti IL-6 therapies under clinical development and there is a lot of research and funding into the IL-6 blocking therapies.

IL-6 is a known muscle atrophy marker and this can be used in future research to look for certain molecules which may induce inflammation and when the presence of IL-6 is removed this may indicate that we have a preventative of muscle inflammation, so we can see how important research into how IL-6 functions with regards to the indication of muscle atrophy along with the IL-6 receptor gp130.

With the results seen with the CCL-5 expression where we saw a decrease in the expression upon a small dose of IL-6, I would like to see future research going into seeing whether we can see an anti-inflammatory effect in certain cytokines especially IL-6. If we were able to find a certain dosage or integration of cytokines which had an anti-inflammatory effect or even a synthesising effect then we could look to rehabilitate muscle or cells within the body, so not only prevent muscle wastage but perhaps help build muscle naturally even after critical illness and ageing.

4.5 Conclusions

Based on our results on the impact of IL-6 on skeletal muscle we have contrasting findings. We see no change in the fibre diameter upon treatment with the cytokine IL-6, which is not what we expected however we have discussed how this may be due to the short incubation time and if possible repeats for the fibre diameter measurements would be done with a longer incubation period of up to a week.

With the TNF-α, CXCL-1 and MURF-1 expressions we see little change with treatment which again may be due to the incubation time or maybe even the concentration of treatment. We also mentioned how these seemingly abnormal findings may be due to the perfect conditions in cell culture and the fact that we may get an increase in TNF-α and MURF-1 may in fact be due to other factors within the bodies system. IL-6 infusion has been shown previously to inhibit TNF-α which in turn keeps MuRF-1 expression down which may be the reason that we see no change in gene expression.

IL-6 and ATROGIN-1 expression does increase as expected upon IL-6 treatment which corresponds to previous research indicating that IL-6 does cause muscle atrophy and damage. However due to the unexpected results with MURF-1 and TNF-α expression it is difficult to say how accurate they are. I feel we should repeat all of the trials with an incubation period of 48 hours so we can compare all of our results having had the same protocol which allows us to make more accurate comparisons.

The CCL-5 expression does increase on a high dose of IL-6, but also decreases on low dosage. I have found evidence for the decrease at low dosage and increase at high dosageThe sensitivity of the CCL-5 molecule is obviously a key factor in its release, as I mentioned earlier is plays a vital role in the proliferation of Natural Killer cells which requires stringent regulation. The time frame is also a crucial role as although we do see an increase in CCL-5 expression we do not see a change in fibre diameter. This is due to the fact that CCL-5 acts as a recruitment molecule for leukocytes into inflammatory site , so when expressed the CCL-5 molecules will not instantaneously have an effect on muscle wasting it will initiate a process which will decrease the muscle fiber diameter, so again the period of time should be increased to see decreased fiber diameter.

Our N number is relatively high for the treatment experiments which increases the accuracy of the results and will therefore decrease the chance of error. I feel further research needs to be carried out on the protocol for the treatment incubation and it should be considered to extend it. The concentrations for the treatment may need to be adjusted for the treatments to show better results, in some cases where we saw no change in the expression I feel the dose should be increased, although this may lead to less clear results for the cytokines and atrophy markers which did show a change at the original doses.

The type of mouse cell line although it seems to have an effect on the results when comparing experiments with similar protocols s