1. Msc Integrated Physiology of Health and
Disease.
By Ketul Desai
27th/08/15
SCHOOL OF MEDICINE AND HEALTH
SCIENCES, UNIVERSITYOF NOTTINGHAM,
QUEENS MEDICAL CENTRE.

2. Table of Contents
The role of ARNT in Cardiac tissue during chronic hypoxia
General Introduction……………………………………………………………………………………………………………1
Literature Review
-Structure andFunctionof HIF 1α and ARNTsubunits……………………………………………………………2
-Regulationof the stabilityof HIF1………………………………………………………………………………………..3
-Gene targetsof the HIF system……………………………………………………………………………………………..5
-Role of HIF 1 in Cardiacdisease……………………………………………………………………………………………..7
-ARNTpotential metabolicrelevance intissue………………………………………………………………………..9
-Therapeuticpotential of HIFsystem……………………………………………………………………………………….10
Materialsandmethods………………………………………………………………………………………………………….14
Results…………………………………………………………………………………………………………………………………20
Discussion…………………………………………………………………………………………………………………………….30
Acknowledgments ………………………………………………………………………………………………………………..40
References…………………………………………………………………………………………………………………………….41

3. Abstract
Background -The heterodimer HIF 1 is a master regulator of oxygen homeostasis. Aryl
hydrocarbon nuclear translocator (ARNT) is part of the basic helix loop helix protein family
(BHLP) and has the ability to heterodimerise with HIF 1α under hypoxia forming the HIF 1
complex. Which in turn can induce transcription of target genes to restore oxygen homeostasis in
tissue. Recent research seems to indicate ARNT may have some important role in cellular
metabolism/organ function. Thus this dissertation will be the first step in to understanding the role
of ARNT in cardiac tissue under chronic hypoxia in an in vivo model. It was hypothesised ARNT
levels will not change in response to chronic hypoxia exposure in cardiac tissue.
Methods and Results- Mice were exposed to chronic hypoxia (11%) in a plexisglass chamber for
three weeks. Western blots were used to analyse levels of the protein ARNT in cardiac and
skeletal muscle tissue in response to chronic hypoxia exposure. Weight of mice was also
analysed prior to incubation into hypoxic chamber and at euthanasia. There was no significant
difference in the levels of ARNT protein in response to chronic hypoxia in either cardiac or
skeletal muscle tissue (P>0.05). However there was a significant difference in the weight of the
mice at euthanasia between normoxic (control) and hypoxic mice (P<0.05).
Conclusions - In conclusion findings of ARNT levels remaining unchanged in response to chronic
hypoxia exposure are consistent with the initial hypothesis.

5. General Introduction
During the course of evolution of multi-cellular organisms, the utilisation of molecular oxygen
was essential for the generation of metabolic energy required for growth and survival. Multi
cellular organisms have developed refined complex networks to be able to adapt in
conditions where oxygen availability is minimal (Brahimi-Horn and Pouysségur, 2007).
Hypoxia inducible factor 1 (HIF 1) is a master regulator of oxygen homeostasis. HIF 1 is a
heterodimeric protein consisting of α and β subunits (Wang et al, 1995). In hypoxic
conditions HIF α is stabilised and dimirises with the βsubunit (ARNT) forming an HIF 1
complex which can bind to the promoter region of targets genes to induce transcription in
attempt to restore oxygen homeostasis, some of which include VEGF, EPO and glycolytic
enzymes (Figure 3) (Neufeld et al., 1999).
Aryl hydrocarbon nuclear translocator (ARNT) also known as hypoxia inducible factor 1β
(HIF 1β), belongs to the basic helix loop helix (bhlh) family of transcription factors and is
constitutively expressed. ARNT mRNA and protein are considered to be maintained at
constant levels irrespective of oxygen availability (Kalio et al 1997). However some recent
hypoxia cell culture based studies have reported findings which suggest ARNT may be
modified under hypoxia. Jiang et al 1997 found ARNT levels within the nuclei of Hep 3B cells
were greatly induced when exposed to hypoxia. Iyer et al 1997 also conveyed similar
findings reporting ARNT levels rising in Hep 3B cell line following exposure to hypoxia.
Furthermore, recent literature based on ARNT seems to suggest it having an important role
in the cellular metabolism of various organs. ARNT gene knockout and siRNA studies
carried out in liver of mice exhibited abnormal glucose tolerance, impaired insulin secretion
and altered islet gene expression mimicking that of a human type 2 diabetic (Gunton et al,

6. 2
2005). In addition, Wu et al, 2014 documented cardiac specific ablation of ARNT leading to
cardiomyopathy with reduced cardiac function which was characterised by the presence of
lipid droplets mediated through PPARα mechanism. These studies indicate ARNT may have
an important role in cellular metabolism/ organ function. Thus this dissertation will be the first
step into understanding the role of ARNT in cardiac tissue under chronic hypoxia in an in
vivo model.
Literature Review
Structure and function of HIF α and ARNT subunits.
The HIF 1 complex is formed when HIF 1 α subunit and the ARNT subunit heterodimerise. Both
subunits are part of the basic helix loop helix (BHLH) / PAS protein family (Wang et al., 1995).
These domains are essential for the interaction and dimerisation of the two subunits (figure 1).
HIF 1 α contains two transactivational domains (TADs) one at the N-terminal of the polypeptide
and another located at the C-terminal (Ruas et al., 2002). These are required for binding at
promoter regions of target genes to induce transcription and are also a site where co activators
such as p300/CBP can bind (Andre et al 2004). Unlike the α-subunit the ARNT subunit contains
only one TAD domain (Figure 1).
Figure 1 (Ke et al 2006) – Illustrates both
HIF 1 α subunit and the constitutively
expressed HIF β subunit structural domains.
Both subunits are part of the BHLH-PAS
protein family. Total number of amino acids
present in each polypeptide is presented in
bold at the C terminal of each respective
subunit of the HIF 1 complex.

7. 3
HIF 1 α is tightly regulated via oxygen concentration. This is due to HIF 1 α containing an oxygen
dependent degradation domain (ODDD) which under conditions where oxygen concentration is
plentiful conserved proline residues at position P402 and P564 are hydroxylated by a family of
prolyl hydroxylase enzymes within the ODDD (Pugh et al., 1997). This targets the α-subunit for
VHL-tumour suppressor mediated ubiquitination and subsequent proteasomal degradation
(Lando et al., 2002). The stability and regulation of the HIF 1 subunits will be explained in more
detail later in the subsequent section of this dissertation.
Regulation of the stability of HIF 1
The ARNT subunit of the heterodimeric HIF complex is considered to be constitutively expressed
within cells and remains stable irrespective of oxygen availability (Kallio et al 1997). However
HIF1 α is tightly regulated via oxygen concentration. Under normoxic conditions, HIF1 α is
hydroxylated by the family of prolyl hydroxylase enzymes at the oxygen dependant degradation
domains (ODDDs) at conserved proline residues 402 and 564. Hydroxylation of HIF1 α leads to
Von hippel lindau ubiquitin E3 ligase complex (PVHL) binding to the αsubunit targeting it for
polyubiquitation. The ubiquitin tags thereby mark the HIF1 α protein for 26s proteosomal
degradation.
Alternatively, under hypoxic conditions HIF1 α is preserved and prevented from being rapidly
degraded via the PHD/PVHL pathway. PHDS require oxygen for its activation therefore under
conditions of low oxygen availability the hydroxylation of HIF1 α doesn’t occur therefore is not
marked by the ubiquitation mechanism for proteosomal degradation (Lando et al, 2002). The
stabilised HIF1 α subunit dimerises with the abundant ARNT subunit within the nucleus forming
the heterodimeric protein HIF 1 complex. The HIF1 complex then induces transcription upon the
association of HREs in the regulatory regions of various genes (figure 2).

8. 4
Figure 2 (Ke et al 2006) – Depicts the mechanism by which HIF α is degraded in
normoxia and by which HIF α is stabilised in hypoxia to form a heterodimer complex.

9. 5
Gene targets of the HIF system.
More than 100 different
downstream gene targets of HIF1
have been identified which are
activated in an attempt to restore
oxygen homeostasis. HIF1
activates the expression of these
genes by binding to a 50- base
pair cis acting HRE located in their
enhancer and promoter regions
(Semenza et al 1991).
Erythropoiesis
The production and maturation of erythrocytes is enhanced in response to hypoxia (Semenza et al
1999). HIF1 mediates increased expression of the hormone erythropoietin (EPO) under hypoxia
which is in turn released from the kidneys and is responsible for erythrocyte formation. Therefore an
increase in the number of erythrocytes within the blood plasma augments the oxygen carrying
capacity of the blood (Semenza et al, 1999).
Furthermore, hypoxia induces HIF1 mediated up regulation of iron metabolising genes which is crucial
for erythrocyte formation. Transferrin is responsible for the transport of Fe3+ into the erythrocytes and
therefore can be rate limiting for erythrocyte production. However under hypoxia transferrin and the
extracellular transferrin receptor which is required for ligand-receptor mediated influx of Fe3+ are also
Figure 3 (Ke et al 2006) – Illustrates some of
the genes which are activated via the HIF 1
complex.

10. 6
upregulated via HIF (Mukhopadhyay et al, 2000). Thereby the enhanced expression of these genes
increases Fe3+ movement into erythroid tissue which is essential for its production and function.
Hence the upregulation of iron metabolising genes optimises erythrocyte production and therefore
increases oxygen carrying capacity of the blood in an attempt to restore oxygen homeostasis at the
site hypoxic tissue.
Angiogenesis
Angiogenesis is process by which new blood vessels form from pre-existing blood vessels.
Under hypoxia the process of angiogenesis is upregulated (Levy et al 1995). Vascular
endothelial growth factor (VEGF) is a potent mediator of angiogenesis. cDNA studies have
demonstrated HIF1 binding sites on VEGF gene promoter regions (Forsythe 1996) thereby
evidently showing the interaction between the HIF1 complex and VEGF gene transcription
under hypoxia. Therefore the induction of VEGF along with other proangiogenic factors would aid
to increase vascular density and hence reduce the diffusion distance of oxygen to hypoxic cells.
Glucose metabolism
In conditions of low oxygen availability, cells are under increased stress to generate energy in the
form of ATP due to a shift in the balance towards glycolysis rather than the more productive
oxygen dependant glucose oxidation. However hypoxic cells elevate their ability to generate ATP
through glycolysis, this is in part achieved via the HIF 1 mediated up regulation of glycolytic
enzymes and glucose transporter proteins (Wenger, 2002). Hypoxia increases the expression of
GLUT 1 and GLUT 3 transporters in hypoxic cells (Chen et al, 2001) therefore enhancing cellular
uptake of glucose allowing for a greater glycolytic potential.

11. 7
In addition most glycolytic enzymes such as phosphofructokinase and pyruvate kinase which are
rate limiting enzymes involved in regulatory steps in the glycolytic pathway (Wong et al 2013).
These enzymes in response to hypoxia have been documented to be upregulated allowing for a
greater potential for glycolytic turnover of glucose (Seagroves et al., 2001).
Role of HIF1 in cardiac disease.
Heart failure is defined as the inability of the heart to maintain its peripheral pumping
requirements. Heart failure has a prevalence of approximately 7 million people in the U.K alone
(British heart foundation, 2015), putting a significant amount of financial burden on the NHS.
In a healthy heart, fatty acids serve as a major fuel substrate for the mammalian heart, providing
~60%-80% of its energy requirements (Opie, 1969). However there is a shift in cardiac fuel
substrate utilisation in the pathophysiological heart (Stanley et al, 2005), which is characterised
by enhanced glycolytic rates of the heart meaning glucose is serving as a major cardiac fuel
substrate as opposed to fatty acids.
Heart failure involves reprograming of cardiac substrate utilisation with decreased fatty acid
metabolism and increased glucose utilisation (Van Bilsen et al, 2004). A similar phenomenon
occurs in hypoxic conditions as it has been previously reported in literature that high altitude
natives such as Himalayan Sherpa’s displayed enhanced glycolytic rates and conversely lower
rates of fatty acid oxidation (Holden et al, 1995). Therefore it can be noted that similar changes in
cardiac substrate metabolism occur in the hypoxic heart and also in the pathophysiological heart.
As the pathophysiological heart can also be exposed to local hypoxia particularly during the
formation of atherosclerotic plaques in lumen of major coronary arteries which can result in
reduced myocardium tissue perfusion (Semenza 2014). This results in a state known as
ischaemia where an adequate amount of O2 is not supplied to cardiac tissue. Thus the changes
in cardiac metabolism are linked in the hypoxic and pathophysiological heart.

12. 8
The shift to increased glucose utilisation and decreased rates of fatty acid oxidation observed in
response to hypoxia in high altitude natives seems to be most likely an adaptive cardiac
response. As glucose is a more of an efficient fuel when compared against fatty acids as
exclusive use of glucose increases the efficiency of ATP production by 12-14%.
The augmented glycolytic rates in the hypoxic heart have been partly attributed to the effects of
HIF 1 stabilisation and hence its transcriptional activity on target genes. The HIF system has
great relevance in cardiac tissue due to cardiac function being heavily reliant on oxygen supply
(Solaro et al 2013). Therefore the maintenance of oxygen homeostasis of heart via the HIF 1
system is crucial.
Kim et al, 2006 reported hypoxia exposure to rats resulted in the HIF 1 mediated up regulation of
pyruvate dehydronase kinase 1 (PDK1) an enzyme responsible for the phosphorylation of
pyruvate dehydronase. Phosphorylation of pyruvate dehydronase leads to its inactivation at the
mitochondrial site thereby reducing the flux acetyl Co-A groups into the mitochondria and hence
decreasing levels oxidative metabolism. In addition, glucose transporter proteins such as GLUT
1/GLUT 4 and glycolytic enzymes such as phosphofructokinase and pyruvate kinase are also
upregulated in response to hypoxia via HIF 1 (Lu et al, 2002), Thereby allowing for augmented
glucose entry and increased glycolytic turnover.
Recent research has documented that PPAR α, a nuclear hormone receptor one of which three
isoforms (α, β, Ω) which are abundantly expressed in heart tissue (Poulsen et al 2012) is a key
regulator of the change observed in cardiac substrate utilisation in the hypoxic heart. As PPAR α
modulates the expression of proteins such as CD36 and β-hydroxyl-acyl-CoA dehydronase (β
HAD) which is a fatty acid transporter protein and a key enzyme involved in the β oxidation of
lipids respectively. PPAR α deficient mice within cardiac tissue specifically express extremely low

13. 9
levels of β HAD and CD36 proteins indicating PPAR αplays an important role in the regulation of
fatty acid metabolism in cardiac tissue (Evans et al 2004).
Under hypoxic conditions, following HIF 1 stabilisation PPAR α is down regulated (Cole et al, in
press) which seems orchestrate the changes in cardiac substrate metabolism. Moreover, the low
levels of β HAD and CPT 1 expression has also been documented to be due to the HIF 1
dependant PPAR α downregulation mechanism. CPT 1 is a vital mitochondrial enzyme which is
essential for the transfer of acyl groups to carnitine allowing the movement of lipids into the
intermembrane space of the mitochondrial which is crucial for the beta oxidation of lipids (Evans
et al., 2004). Thus it clear that HIF 1 activation by hypoxia can orchestrate vast changes to
cardiac substrate metabolism through the interaction of the nuclear hormone receptor PPAR α.
ARNT potential metabolic relevance in tissue.
Aryl hydrocarbon nuclear translocator (ARNT) forms a heterodimer with HIF 1α in response to
hypoxia mediating the transcription of target genes therefore is an essential component of the
HIF 1 complex mediated effects within cardiac tissue. Recent literature seems to suggest that the
ARNT subunit may also have an important role in the cellular metabolism of various organs.
Interestingly humans who suffer from type 2 diabetes, ARNT expression levels within the
pancreas and liver were discovered to be extremely low (Wang et al 2009). In addition ARNT
deletion studies in mice resulted in a condition which mimicked that of a type 2 diabetic, thus
indicating ARNT has some metabolic role within tissue. ARNT may also have metabolic
relevance within cardiac tissue as Wu et al 2014 documented cardiac specific ablation of ARNT
resulted in cardiomyopathy characterised by triglyceride accumulation mediated through PPAR α
.

14. 10
Therefore as hypoxia is an integral component of heart disease and recent literature seeming to
suggest ARNT having some potential role in cardiac function. This dissertation will be the first to
explore the role of ARNT in cardiac tissue under chronic hypoxia in an in vivo model, as there is
currently a lack of literature based on phenomena.
Therapeutic potential of HIF in cardiac disease.
The HIF facilitated effects which occur in response to hypoxia provide potential therapeutic
targets which can be exploited. During ischaemic heart disease (IHD) promoting the effects of
HIF can be advantageous (Vincent et al, 2000). As ischaemic diseases can be characterised by
atherosclerotic plaques which are deposited in the lumen of major coronary arteries which
ultimately results in reduced myocardium tissue perfusion, hence localised tissue hypoxia
(Semenza 2014). Under these circumstances in a healthy individual the normal physiological
response is the hypoxia induced activation of HIF 1 system. This ultimately results in the
transcription initiation of vascular endothelial growth factor (VEGF). VEGF is a potent initiator of
the vascularisation of new blood vessels resulting in increased perfusion to hypoxic tissue
(Vincent et al, 2000).
However in severe cases of IHD the activation of the HIF 1 system in response to hypoxia is
dampened therefore as a result the transcription of VEGF is reduced leading to cardiac
complications (Semenza 2001). Consequently under these circumstances up regulating the
activity of HIF 1 to induce VEGF transcription would be beneficial in patients with severe IHD as

15. 11
capillary density around hypoxic myocardium tissue will be increased reducing the diffusion
distance of oxygen.
Furthermore the importance of HIF and hence VEGF transcription is highlighted in HIF1α
knockout mice studies, as it was discovered that failure to maintain VEGF expression and thus
vascularisation of new blood vessel lead to accelerated onset of myocardial infarct in an
experimental setting (Sano et al 2007).
One method in which the process of angiogenesis can be upregulated is through the inhibition of
prolyl hydroxylases enzymes. Prolyl hydroxylases (PHDS) are members of the iron and 2
oxoglutarate dependent dioxygenase enzymes and are responsible for the ubiquitin/PVHL
mediated degradation of HIF 1α in normoxia thereby preventing HIF 1 stabilisation. Inhibiting
theses enzymes in cardiovascular disease particularly has recently gained a lot of interest at the
clinical level and many pharmaceutical companies are carrying out research in the hope of
finding a novel therapeutic drug (Selvaraju et al 2014).
As it is already known HIF 1α degradation is reliant on the oxygen dependant activation of PHD
therefore preventing HIF 1 stabilisation and thus transcription of proangiogenic factors such as
VEGF. Under these conditions HIF 1 stabilisation can be valuable and can be achieved through
the inhibition of PHD enzymes. GlaxoSmithKline has recently assessed the efficacy of their PHD
inhibitor termed GSK360A on a rat myocardial infarct model. It was discovered those rats treated

16. 12
with GSK360A maintained cardiac function and of more significance there was a two fold
increase in the microvasculature on the infarct zone (Bao et al 2010). Thus highlighting the
potential PHD inhibitors can have on treating cardiovascular diseases however far more testing
is required at the clinical level.
It is well documented in literature that in a pathogenic heart there are significant changes to
cardiac substrate utilisation. With a greater reliance on enhanced glycolytic rates rather than the
oxidation of lipids (Burkart et al., 2007). The changes observed in fatty acid metabolism of the
heart has been suggested to be regulated via the nuclear hormone receptors PPAR. Of the three
isoforms (α, β, Ω) PPAR α and PPAR Ω are expressed at relatively high levels in cardiac tissue
and display large overlap in gene targets (Poulsen et al, 2012). Mice with cardiac specific
overexpression of the nuclear hormone receptor PPAR α displayed high levels of free fatty acid
(FFA) uptake, triglyceride accumulation and reduced levels of glucose oxidation ultimately
leading to cardiomyopathy and hypertrophy (Burkart et al, 2007). Conversely, in hypoxia HIF
dependant downregulation of PPAR α prevented cardiac metabolic reprogramming (Cole et al, in
press), thus signifying the importance of the role PPARs play in cardiac substrate metabolism
which can be of potential therapeutic value in the future with further testing.
From these studies it seems to suggest that in HIF plays a protective proangiogenic role in
response to some cases of IHD and potential pathogenic role in the reprogramming of cardiac

17. 13
metabolism (Semenza, 2014)particularly in end stage heart failure resulting in energetic failure.
Thus the complexity of HIF mediated adaptive effects are highlighted.
Interestingly HIF 2 another member of the HIF family can also heterodimirise with ARNT inducing
transcription of target genes however these largely target only proangiogenic factors such as
VEGF (Bai et al., 2008). Moreover HIF 1 seems to largely influence genes such as PDK 1 which
is largely responsible for reprogramming glucose metabolism (Kim et al, 2006). Thus further
research for potential therapies is required into looking at mechanisms in to whether if possible,
to selectively down regulate HIF 1 and up regulate HIF 2 activity in the failing heart.

18. 14
Materials and Methods.
Animals
4 week old male CD1 mice (n=14) were housed on a 12 hour light/dark cycle and fed ad libitum.
All procedures carried out confirmed with the ethical guidelines of the U.K. home office and the
Animal scientific procedures Act (ASPA) 1996 legislation.
Chronic Hypoxic housing
Mice were initially exposed to a seven day hypoxic acclimatisation period, during which
plexiglass chamber oxygen content was lowered on a daily basis, 2% per day down to 15% O2
then 1% per day until a minimum of 11%. Thereafter oxygen level was maintained at11% for 12
days. Mice were weighted on a daily basis, involving a brief period of normoxia (15 seconds).
Following hypoxic housing, all mice exposed to 1 hour of room air, to discount short term effects
of re-oxygenation on cardiac function. Thus all results represent long term adaptation to hypoxic.
A

19. 15
B
Figure4A/B - (A) Illustrates the hypoxic chamber the mice were incubated in (B) acclimatisation period and the
hypoxic exposure protocol.
Buffers
Cytosolic protein extraction buffer includes 50mM Tris-HCL, pH 7.5, 1 mM EDTA, 1mM EGTA,
1% IGEPAL, and 0.1% 2-mercaptoethanol.
10x electrophoresis buffer contained 250nM Tris, 1.92M glycine, 1% SDS this was diluted down
to a 1x solution with deionised water during gel runs. 4x resolving gel buffer contained the
following 1.5M Tris-HCL, PH 8.8, 0.4% SDS. 4x stacking gel buffer contains 0.5M Tris-HCL, PH
6.8, 0.4% SDS. Transfer buffer contained the following 25mM Tris, 192mM glycine, 20%
0
5
10
15
20
25
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
%OFOXYGEN
DAYS IN THE CHAMBER

20. 16
methanol. 10x Tris buffered saline (TBS) contained 100mM Tris- HCL pH 7.6, 1.5M NaCl, TBS-T
contains 1x TBS with 0.1% tween.
Primary and secondary antibodies
The primary ARNT antibody specific to the nuclear protein ARNT was raised in rabbit species
and purchased from Cell signalling Technology Co.
The primary actin antibody was provided as a 30 µg/ml aliquots by Kevin bailey at the university
of Nottingham medical school.
The secondary antibody which was used was the following - Polyclonal swine Anti-rabbit
immunoglobulins purchased from Dakocytomation.co.
Tissue collection
After the three week hypoxia exposure period the mice were removed from the hypoxic chamber
and euthanased with a perinatal injection of sodium pentobarbital (30mg/kg) into the abdomen.
Cardiac tissue and skeletal muscle tissue were taken and immediately immersed in liquid
nitrogen to freeze tissue to avoid any modifications induced by tissue hypoxia samples were then
subsequently stored at -800c.

21. 17
Cell lysate preparation
During the homogenisation process 60mg of frozen cardiac and skeletal muscle tissue was
weighted and homogenised in 600µl of cytosolic buffer with the addition of 6µl protease inhibitor.
Lysates were then centrifuged at 13,000g for 10 minutes at 4oc with the supernatant being
saved. Total tissue protein content was quantified via a BSA standard curve protein assay using
bovine serum albumin as a protein standard. This assay utilises a set of gradually increasing
known protein concentration standards. Known standards are read at a wavelength of 582nm on
a spectrophotometer along with samples of unknown protein concentration in a 96 well plate.
Absorbance values for the standards will produce a standard curve and the equation of the
standard curve can be used in order to calculate the protein concentration of unknown samples.
Western blot analysis
Cardiac and skeletal muscle cell lysate samples were standardised to 5mg/ml whilst using
deionised water as a diluent. Prior to running the samples sample buffer was added at a 2:1 ratio
along with 3% DTT weight by volume. Subsequently the samples were heated for 5 minutes and
ran on polyacrylamide gels (30% SDS-PAGE) for 2 hours at 50 milliamps constant current. The
samples were then transferred to a Polyvinylidene Difluoride (PVDF Amersham) membrane at
constant current 40 miliamps and left overnight. The following morning transfer efficiency of the
proteins to the PVDF membrane was checked via the use of ponceau stain. After this samples

22. 18
were washed in deionised water for 5 minutes to remove any excess ponceau dye. Sample were
then left in 5% BSA TBS-T blocking solution for one hour with gentle shaking. The samples were
then incubated with the primary antibody which is specific to the protein ARNT in 5% BSA TBST
solution at 4oc with a 1:1000 dilution and left overnight with gentle shaking.
The next morning the PVDF membranes containing the samples were washed 5 times for 5
minutes each with TBST. Following the washes the samples were incubated with the secondary
antibody in 1% BSA - TBST solution at a 1:2000 dilution for one hour with gentle shaking. The
washes of the membranes with TBST were carried again as previously mentioned.
Subsequently, the samples were developed using an enhanced chemiluminence kit (Amersham
horseradish peroxide) and exposed for 5 minutes in the Fuijifilm LAS - 3000 mini to observe the
protein bands present. Multiple exposures were carried out to ensure linearity.
Quantification of ARNT
In order to calculate actual concentrations of ARNT in the cardiac and skeletal muscle samples.
A BSA assay was used in order to calculate protein concentration of cell lystates and hence load
a known concentrations of protein (i.e. 5mg/ml - cell lysates) into respective lanes and hence
resolved via SDS-PAGE. Western blots were then developed via the ECL technique ensuring
linearity with multiple exposures. Protein bands were quantified via the use of ImageJ computer

23. 19
software programme in order to calculate the relative density of each band and to calculate
protein concentration present.
Statistical analysis
All statistical analysis and significance testing was carried out on the computer software
programme IBM SPSS. Two way repeated measures ANOVA and a two tailed t test were
performed with significance tested at P (≤0.05).

24. 20
Results
Reproducibility of western blot technique.
Western blot of β actin from mouse skeletal muscle tissue presented to demonstrate competence of
the technique. 5mg/ml of skeletal muscle protein cell lysate were loaded into lane 1 of gels and serial
dilution were carried out for the remaining respective lanes.
Final protein concentration loaded into each respective lane after accounting for sample buffer was
calculated and lane 1 came to a concentration of 3.3mg/ml.
β actin
A
B
0.00
0.20
0.40
0.60
0.80
1.00
1.20
3.3 1.6 0.83 0.41 0.21 0.12
Relativedensity
(µg/ml)
Final concentration of Actin
(mg/ml)
Figure 5 – Depiction of the reproducibility of the western blot technique. (A) Illustrates the western blot at
serial dilutions of actin (n=3) with the highest concentration at 3.3mg/ml (Lane 1) which is represented
via darkest band (figure4A). (B) Band intensities presented as fold change relative to lane 1.

25. 21
3.2. Transfer efficiency of samples
In order to measure the efficiency of the transfer process a Ponciea stain was used to visually
observe the presence of protein bands being transferred from the gel to the PVDF membrane.
Ponciea stains from both Cardiac (A) and skeletal muscle (B) are presented with either noromoxic or
hypoxic samples loaded in alternating fashion for e.g. (N1, H1 etc.)
ARNT has a molecular weight of 87kD therefore the bands present just above the 75 kD molecular
weight region is where ARNT was expected to be located.
Figure 6 – Demonstrates Ponciea stains of normoxic (N1-7) and hypoxic (H1-7) samples loaded onto
gels in alternating fashion (for e.g. N1,H1 etc.) from either cardiac (A) and skeletal muscle tissue (B)
Blue arrow indicates the molecular weight region of where typically the protein ARNT (87kD) is
located.
Aii
Aii
B Bii
N1 H1 N2 H2 N3 H3 N4 H4
N5 H5 N6 H6 N7 H7
N1 H1 N2 H2 N3 H3 N4 H4 N5 H5 N6 H6 N7 H7
75
kD
A

26. 22
Figure 7 demonstrates a measure of transfer efficiency of cardiac and skeletal muscle tissue
as well as equal loading of samples.
Cardiac samples demonstrate efficient transfer of proteins from gels to PVDF membrane as
their relative densities were close to that of the control. Furthermore there is little variation
between normoxic (+/- 0.14) and hypoxic (+/-0.11) cardiac samples indicating competence of
the technique. In addition statistical analysis of variation between conditions for cardiac
samples indicated little variance (P>0.05).
Skeletal muscle samples demonstrates good transfer of protein from gels to PVDF
membrane also as relative densities are close to that of the control. Statistical analysis of the
variation for the transfer of protein between conditions (hypoxic vs normoxic) for skeletal
muscle samples was not significant (P>0.05).
However in comparison to the cardiac samples it seems as if skeletal muscle transfer is
more variable due to greater standard deviations of normoxic (+/-0.31) and hypoxic (+/-0.24)
samples relative to that of cardiac samples (+/-0.14 and +/-0.11) respectively. Furthermore
statistical analysis of variation between cardiac and skeletal muscle samples was statistically
significant (P<0.05).

27. 23
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Relativedensityof
poncieusstains
Normoxia Hypoxia Normoxia HypoxiaControl
Skeletal muscle samplesCardiac samples
Figure 7- Quantified poncieu stain blots from the molecular weight region of 100 -75kD as the ARNT
protein (87kD) is located within this range. Data presented as mean (+/-) of four independent
experiments (n=4). Cardiac and skeletal muscle samples have been normalised against lane 1 of their
respective gels and presented as the control.

28. 24
B
Immunoblotting for the protein ARNT.
During immunoblotting for ARNT certain problems were encountered. One of which was the presence
of a nonspecific background noise on the blots (figure 8A).
After a series of experiments carried out to troubleshoot this particular problem. It was identified to be
due to large amounts of nonspecific binding by the secondary antibody. The secondary antibody
incubation dilution was therefore adjusted from a 1:1000 (figure 8A) to a 1:2000 (figure 8B) and
evidently reduced the amount of background noise (Figure 8).
Figure 8 – Illustrates a summary of the optimisation process of reducing the background noise on the
immunoblots of ARNT. (A) Immunoblot for ARNT in cardiac tissue from normoxic (N1-4) or hypoxic
(H1-4) samples with secondary antibody incubation at 1:1000. (B) Optimised immunoblot of ARNT in
cardiac tissue with secondary antibody incubated at 1:2000 dilution.
ARNT has a molecular weight of 87kD. However there are many other proteins which also exist at this
particular molecular weight such as tropomyosin, nesprin etc which are also abundantly present in
mouse cardiac and skeletal muscle. Hence signal could have been due to nonspecific binding.
A N1 H1 N2 H2 N3 H3 N4 H4 N1 H1 N2 H2 N3 H3 N4 H4

29. 25
Therefore to be entirely sure of the protein bands observed within the range of 100kD – 75kD to be
the target protein ARNT a positive control from a HEP G2 cell line was run to confirm this.
Figure 9 – Confirmation of ARNT immunoblots. (A) Demonstrates protein bands of ARNT from (A-
Normoxic cardiac, B–Hypoxic cardiac, C- Normoxic skeletal muscle, D- Hypoxic skeletal muscle, E- Normoxic
cardiac and F- Hypoxic cardiac) samples which typically has a molecular weight of 87kD. Positive ARNT
control lane from HEP G2 cell line confirms the detection of ARNT within cardiac and skeletal muscle
tissue. (B) A positive ARNT control immunoblot carried out by cell signalling techonology.co which is
the company where the primary ARNT antibody was purchased from.
100
kD
75
kD
50
kD
MW
(kD)
Positive ARNT
control in HEP
G2 cell line.
A B
A B C D E F

30. 26
31.00
32.00
33.00
34.00
35.00
36.00
37.00
0 5 10 15 20
Weight
(g)
Time in chamber
(Days)
Normoxia
Hypoxia
Analysis of weight change.
The effect of O2 concentration on body weight of the mice was also analysed over a period of
three weeks. At the point of incubation into the plexisglass chamber mice exposed to O2
concentration of 11% (Hypoxia) had a mean weight of 32.9 g and mice exposed to O2
concentration of 21% (normoxia) weighted 32.5 g. The weight of the mice was checked
regularly and evidently it can be observed weight of mice gradually increased in both
conditions. However there is a large difference between mean weights at the point of
euthanasia which occurred at the end of the three week period (Hypoxia -33.46) and
(Normoxia – 35.94) respectively.
Figure 10– Illustrates the changes in weight (g) of the mice during hypoxia and normoxia in
the plexisglass chamber. Normoxia mice (n=7) were exposed to 21% atmospheric oxygen
for three weeks and hypoxic mice (n=7) exposed to 11% O2 for three weeks also. Statistical
analysis was carried out using two way repeated measure ANOVA with significance tested
at (P0.05). A statistical difference was seen between the weight of mice in the normoxic
condition compared to the weight of mice in the hypoxic condition (P<0.05).
Point of
euthanasia

31. 27

32. 28
The effect of chronic hypoxia on cardiac ARNT expression.
In order to investigate whether chronic hypoxia altered ARNT expression in CD1 mouse cardiac
tissue. A 3 week protocol that began with a 7 day lowering of ambient oxygen concentration from 21%
- 11% in daily increments to produce physiological hypoxia (figure 11A). Samples from either
normoxic (N) or hypoxic (H) conditions were loaded into respective lanes in alternating fashion for e.g.
N1, H1.
CD1 cardiac tissue in normoxia exhibited a mean ARNT relative density of 1.20 S.D (+/-0.23).
Interestingly in hypoxic condition, mean ARNT relative density appears to be lower 0.86 S.D (+/-0.31)
compared to normoxic condition. However this difference is not statistically significant (P>0.05).
N1 H1 N2 H2 N3 H3 N4 H4 N5 H5 N6 H6 N7 H7
β actin β actin
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
Normoxia (control)
RelativedensityofARNT
(foldchange)
Hypoxia
A
B
Figure 11– Western blot analysis of ARNT in 4 week old CD1 mice cardiac tissue. (A) Western blots of ARNT
from either normoxic (N1-7) or hypoxic (H1-7) conditions. Housekeeping protein β actin used as a loading
control. (B) Graph represents quantified ARNT blots of mean +/- SD of (n=3) independent experiments. A
two tailed t test was performed and a significant difference was not observed between normoxic and hypoxic
conditions for the cardiac samples (P>0.05).

33. 29
The effect of chronic hypoxia on skeletal muscle ARNT expression.
Skeletal muscle ARNT expression exhibited a mean relative density of 1.25 S.D (+/-0.22). Likewise
with cardiac tissue skeletal muscle ARNT expression appears to be lower with a mean relative density
of 1.06 S.D (+/-0.3) (figure 12B).
However similar to CD1 cardiac tissue there was not statistical significant difference (P>0.05)
between chronic hypoxia and normoxia conditions in skeletal muscle.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Normoxia
RelativedensityofARNT
(foldchange)
Hypoxia
A
B
N1 H1 N2 H2 N3 H3 N4 H4 N5 H5 N6 H6 N7 H7
Figure 12 – Western blot analysis of ARNT expression in 4 week old CD1 skeletal muscle tissue. (A) Western
blots of ARNT from either normoxia (N1-7) or hypoxia (H1-7). Due to lack of time blots for β actin as a loading
control were not carried out for skeletal muscle (B) Graph represents quantified ARNT blots of mean S.D (+/-)
of (n=3) independent experiments. A two tailed t test was performed and a significant difference was not
observed between normoxic and hypoxic conditions for skeletal muscle samples (P>0.05).

34. 30
Discussion
Previous studies on the protein ARNT have seemed to suggest ARNT having an important
cellular metabolic role. As low levels of the protein ARNT were observed in the pancreas and
liver of human subjects suffering from type 2 diabetes (Wang et al 2009). In addition ARNT
deletion studies in mice liver and heart exhibited symptoms of abnormal metabolism which
mimicked that of a diabetic (Gunton et al 2005) and (Wu et al 2014). Hence these studies
imply ARNT has some metabolic role.
Hypoxia is defined as a diminished availability of oxygen to bodily tissue and is an integral
component of cardiovascular disease (Simon et al 2008). Particularly in cardiac ischaemia,
as the formation of atherosclerotic plaques in lumen of coronary arteries can reduce
myocardium tissue perfusion resulting in local tissue hypoxia (Semenza et al 2014). Thus
hypoxia has clinical relevance to cardiovascular disease.
However previous literature regarding the molecular physiology of ARNT in vivo hypoxia has
not been consistent with some of the findings from studies using cell based experimental
models. Hep 3B is a cell line derived from human liver tissue and has previously been used
as an experimental model in hypoxia studies. Jiang et al 1997, reported ARNT protein levels
were greatly induced in the nuclei of Hep 3B cells when exposed to 1% O2 as observed on
immunoblots derived from nuclear lysate extracts. In support of these findings Iyer et al

35. 31
1997, reported consistent findings using the same cell line and hypoxia exposure (1% O2).
Clearly these studies suggest increased levels of ARNT protein expression in response to
hypoxia.
The observations made in these studies are not consistent with previous literature regarding
the molecular physiology of the ARNT/ HIF 1α subunits. As it is generally believed that only
HIF 1 α concentrations is regulated via oxygen concentration through the PHD/PVHL
degradation pathway (Kamura et al 2000). ARNT is believed to be ubiquitously expressed
and remain constant irrespective of oxygen concentration (Kim et al 2004).
Therefore as hypoxia is an integral component of cardiovascular disease and cell culture
studies measuring ARNT are not consistent with the previous molecular physiology of
hypoxia. This dissertation will be the first to explore the role of ARNT in cardiac tissue under
chronic hypoxia in an in vivo model.
In order to carry out the western blot technique on the protein ARNT it is was first important
to be entirely competent with the technique so that valuable resources such as tissue and
antibody are not wasted. Therefore many practice runs on the abundantly expressed
housekeeping protein βactin was run to ensure this. Figure 5 represents serial dilutions of β
actin conducted from three independent runs. From figure 5 it can be observed that the
standard deviation errors bars at each concentration of actin are generally small. This

36. 32
indicates a high precision and thus reproducibility of the western blot technique (Cumming et
al 2007).
Poncieu staining was utilised in order to measure transfer efficiency of proteins from gels to
the PVDF membrane and also to determine equal loading of samples. Data particularly from
cardiac samples seem to indicate efficient transfer and equal loading of protein as samples
from both normoxic (1.13 +/- 0.14) and hypoxic conditions (1.06+/- 0.11) have relative
densities close to the control (1). Furthermore the low standard deviation error bars indicate
high precision within the data (Cumming et al 2007).
Looking at the skeletal muscle samples in figure 7 the transfer of protein seems to be more
variable compared to that of cardiac samples. This means there is less confidence in the
data for the transfer of protein for skeletal muscle samples compared to cardiac. The reason
for increased variability in the transfer of skeletal muscle tissue may be due to differences
encountered during tissue and cell lysate preparation of the two different types of tissue.
During the western blot technique it is important to validate the protein bands observed on
blots and to be entirely sure of the bands detected to be the target protein. This is important
because there are also many others proteins which also exist at the expected molecular
weight range of the target protein (Marx 2013). Therefore there is a chance of nonspecific
binding via antibodies to proteins other than the target protein. Figure 9 provides

37. 33
confirmation that the protein bands obtained from cardiac and skeletal muscle from both
hypoxic or normoxic conditions is indeed the target protein ARNT. As the bands are present
within the molecular weight range of 75-100kD which is typically where ARNT (87kD) would
be located. In addition the positive control run in Hep G2 cell line confirms the presence of
ARNT in the same region as the cardiac and skeletal muscle samples.
Exposure to chronic hypoxia can have drastic effects on weight loss. Referring to figure 10,
it can be observed that mice only in the normoxic condition gained weight over the three
week protocol. As the weight of the hypoxic mice at euthanasia (33.5g) is very similar to that
of baseline (33.1g). Contrastingly the normoxic control mice continued to gain weight and
show vast differences in weight at euthanasia (36.1g) compared to baseline (32.5 g).
Evidently from figure 10 it is clear that oxygen availability can have an impact on the
maintenance of weight. Interestingly this finding is consistent with other studies which have
been presented in literature and seem to claim skeletal muscle atrophy is a key factor. Seen
as skeletal muscle accounts for a significant amount of total body mass, it is fair to say
skeletal muscle atrophy can have a significant impact on total body mass. In support of this
claim Hoppeler et al 1999 reported significant skeletal muscle atrophy in response to 40
days of chronic hypoxia exposure in human Himalayan expedition subjects which was
reflected in total weight lost. Furthermore Mac Dougual et al 1991 studied skeletal muscle

38. 34
fibre atrophy in active climbers and stated that muscle fibre atrophy is unavoidable at
altitudes above 16,000 feet and is independent of activity level. This has some relevance to
the current study as mice were exposed to 11% oxygen and this is representative of
altitudes as a high 17,000 feet.
The mechanisms regulating muscle mass during exposure to high altitude and thus low O2
tension are poorly understood. However in vivo animal studies give insight to anabolic and
catabolic process in response to chronic hypoxia (Chaudhary et al 2012). Favier et al 2010,
reported rats exposed to hypoxia equivalent of 18,000 feet for 3 weeks resulted 15%
decrease muscle mass. Furthermore rates of protein synthesis and protein degradation were
also observed via radioactively labelled amino acid techniques. It was concluded that rates
of protein degradation were 5 fold higher than that of protein synthesis which resulted in
muscle loss. As the maintenance of skeletal muscle mass is largely dependent on the
balance between rates of muscle protein synthesis and muscle protein breakdown (Murton
et al 2010) a change in the balance between them can have an impact on muscle mass.
Therefore these studies provides potential insight into mechanisms by which muscle mass is
lost in response to hypoxia.
The current study demonstrates for the first time there is no significant difference (P>0.05) in
the expression of the protein ARNT within cardiac tissue in an in vivo model in response to

39. 35
hypoxia (figure 11b). Although it was not expected for ARNT levels to change in response to
hypoxia as it is generally believed to be expressed ubiquitously within the nucleus and is not
regulated via oxygen tension (Maxwell et al 1999).
However previous findings from cell culture based studies report inconsistent findings which
does not match the molecular physiology of the HIF system in response to hypoxia. As Jiang
et al 1997 reported ARNT levels were greatly induced in response to hypoxic exposure (1%
O2) in Hep G2 cell line. Furthermore, some of the in vivo studies within literature based on
this area seem to suggest an organ specific regulation of ARNT. Stroka et al 2001, reported
ARNT levels remained constant irrespective of the hypoxic condition in both brain and
kidney tissue, however liver tissue displayed a significant increase which was contaminant
with rising HIF 1α concentrations in response to hypoxia. In addition to Strokas study a
reoxygenation protocol was also carried out on all tissue and only HIF 1 α was rapidly
degraded in response to rising O2 concentration with ARNT levels remaining unchanged.
Therefore it may well be ARNT expression is controlled on an organ specific level however it
is important to highlight the differences in the hypoxia exposure protocol between the current
study and Strokas study. Firstly Stroka exposed mice to 6% oxygen for a total of 12 hours
whereas in the current study mice were exposed to 11% oxygen for a total of three weeks.
From this one may state that Strokas protocol is more extreme than the current study as 6%

40. 36
oxygen exposure is representative of altitudes as high as Mount Everest (Osculati et al
2015) and therefore may induce drastic adaptations within tissue as opposed to a more
chronic hypoxia exposure protocol seen in the current study. Thus differences in findings
may be explained via different hypoxic exposure protocols. However further studies are
required using in vivo models with identical hypoxia exposure protocols on differing tissue in
order to allow comparison and confirm this. Regardless the current study states that in
cardiac tissue specifically ARNT expression does not alter significantly under conditions of
chronic hypoxia (11%).
The effect of chronic hypoxia on the expression of ARNT was also analysed in skeletal
muscle to observe any potential differences on the levels of ARNT expression between two
different types of muscle (i.e. cardiac and skeletal). Similarly to cardiac tissue the current
study identified no significant difference on the levels of ARNT expression in skeletal muscle
between normoxic and hypoxic conditions (P>0.05).
In exercising muscle local oxygen tension falls considerably which often can result in local
tissue hypoxia (Gustafsson et al 1999). Under these conditions it was observed that
expression of ARNT mRNA and HIF 1α mRNA were significantly induced leading increased
expression of the potent proangiogeneic factor VEGF through the HIF complex. This would

41. 37
result in increased capillary density around the exercising muscle and reduce diffusion
distance of oxygen so that oxygen homeostasis can be maintained.
In the current study mice were exposed to chronic hypoxia lasting a total of 21days at 11%
oxygen. During exercise it is unlikely muscle will experience chronic hypoxia of this
magnitude and hence it is more likely that exercising muscle is exposed to periods of acute
hypoxia as opposed to chronic (Romer et al 2006). Thus the differences in adaptation of
skeletal muscle to chronic and acute hypoxia are highlighted here.
Relatively recent research seems to suggest hypoxia may be of some relevance to the
dysregulated metabolic states which are commonly associated with obesity. Obesity poses
as a major epidemic worldwide with current estimates of international obesity reaching 312
million (James et al 2004). Obesity is defined as the abnormal accumulation of body fat
usually characterised by a BMI > 30kg/m2. Dysregulated lipid and glucose metabolism is
frequently associated with obese patients (Redinger 2007). Ectopic fat accumulation
particularly at the site of the liver and pancreas has been shown to increase the chance of
insulin resistance (Snel et al 2012). Recent literature documents hypoxia has some
relevance to the occurrence of dysregulated metabolism observed in the obese. Adipose
tissue is specialised for the storage of fats however in obesity the rate of expanding adipose
tissue cannot be matched by the oxygen supply resulting in relative hypoxia within adipose

42. 38
tissue (Jiang et al 2011). Given that hypoxia mediated effects are mainly regulated via
hypoxia inducible factor 1 (HIF 1) and all isoforms of the HIF system is expressed in adipose
tissue widely (Hatanaka et al 2009). It has been suggested HIF dependant responses to
hypoxia may contribute to the dysregulated lipid and glucose metabolism observed in obese
patients.
Jiang et al 2011 fed mice a high fat diet (HFD) for 12 weeks to induce obesity and
determined their metabolic phenotypes. Mice which were deficient in ARNT and HIF 1α
displayed improvements in glucose tolerance and insulin resistance. This was indicated via
increased phosphorylation of the protein Akt which is essential for insulin stimulated glucose
uptake and has been shown to be underactive in insulin resistance (Welsh et al 2005).
Furthermore adipocyte specific ARNT and HIF 1α null mice were resistant to HFD induced
weight gain. Therefore it seems ARNT/HIF 1αand thus the HIF 1 complex may contribute to
insulin resistance and weight gain.
One potential mechanism by which HIF 1 may contribute to insulin resistance in adipocytes
is through the expression of the protein SOCS3 (Jiang et al 2011). SOCS3 protein has
previously been documented to bind to phosphorylated tyrosine 960 of the insulin receptor
and inhibit its auto phosphorylation (Senn et al 2003). In addition SOCS3 deficiency
increases insulin stimulated glucose uptake in adipocytes (Shi et al 2004). Therefore

43. 39
decreased SOCS3 protein expression in ARNT and HIF 1α null mice may explain the
improved insulin signalling observed in these mice.
Clearly it can be observed HIF mediated effects under hypoxia within adipocytes contributes
to the changes in glucose and lipid metabolism seen in the obese. In the current study, if
time and fewer technical problems had allowed the next step was to examine whether mice
fed a high fat diet in combination with hypoxia had abnormal levels of ARNT. Thus future
work should focus on the effect of a high fat diet induced obesity and in combination with
chronic hypoxia to investigate levels of ARNT expression within different tissue. As this will
allow comparisons to be made on the levels of ARNT expression between HFD induced
obesity in combination with chronic hypoxia and chronic hypoxia alone.
In summary the main findings of the current study are, that there is no significant difference
between normoxic and hypoxic conditions on the level of ARNT expression in either cardiac
or skeletal muscle tissue.

44. 40
Acknowledgements
This dissertation could not have been completed without the guidance of my project
supervisor Dr M Cole at the University of Nottingham. I would also like to thank Kevin Bailey
for his continued guidance and support throughout the course of the project particularly
during the practical element.
Finally I would like to extend my appreciation to my family for their constant support and
love.

45. 41
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