Msc Integrated Physiology of Health and Disease.

By Ketul Desai27th/08/15

SCHOOL OF MEDICINE AND HEALTH SCIENCES, UNIVERSITY OF NOTTINGHAM, QUEENS MEDICAL CENTRE.

Table of ContentsThe role of ARNT in Cardiac tissue during chronic

hypoxiaGeneral Introduction……………………………………………………………………………………………………………1

Literature Review-Structure and Function of HIF 1α and ARNT subunits……………………………………………………………2

-Regulation of the stability of HIF 1………………………………………………………………………………………..3

-Gene targets of the HIF system……………………………………………………………………………………………..5

-Role of HIF 1 in Cardiac disease……………………………………………………………………………………………..7

-ARNT potential metabolic relevance in tissue………………………………………………………………………..9

-Therapeutic potential of HIF system……………………………………………………………………………………….10

Materials and methods………………………………………………………………………………………………………….14

Results…………………………………………………………………………………………………………………………………20

Discussion…………………………………………………………………………………………………………………………….30

Acknowledgments ………………………………………………………………………………………………………………..40

References…………………………………………………………………………………………………………………………….41

AbstractBackground -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.

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, 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).

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.

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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.

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).

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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

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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.

Figure 3 (Ke et al 2006) – Illustrates some of the genes which are activated via the HIF 1 complex.

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 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.

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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.

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%.

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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 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.

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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.

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

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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 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 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.

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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 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.

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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.

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A

12

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% 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.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 210

5

10

15

20

25

Days in the chamber

% o

f Oxy

gen

Acclimatisation period

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.

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

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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

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 software programme

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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).

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.

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3.2. Transfer efficiency of samplesIn 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.

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β actin

B

Aii

B Bii

N1 H1 N2 H2 N3 H3 N4 H4 N5 H5 N6 H6 N7 H7

75kD

A

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.

A

3.3 1.6 0.83 0.41 0.21 0.120.00

0.20

0.40

0.60

0.80

1.00

1.20

Final concentration of Actin (mg/ml)

Rela

tive

dens

ity

(µg/

ml)

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. 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).

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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).

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0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6Re

lativ

e de

nsity

of

ponc

ieus

sta

ins

Normoxia Hypoxia Normoxia HypoxiaControl

Skeletal muscle samplesCardiac samples

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.

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

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A B C D E F Positive ARNT control in HEP G2 cell line.

A B

N1 H1 N2 H2 N3 H3 N4 H4 N1 H1 N2 H2 N3 H3 N4 H4

100 kD75 kD

50 kD

MW (kD)

A

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.

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.

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Point of euthanasia

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).

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0 5 10 15 2030.00

31.00

32.00

33.00

34.00

35.00

36.00

37.00

NormoxiaHypoxia

Time in chamber(Days)

Wei

ght

(g)

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).

23

N1 H1 N2 H2 N3 H3 N4 H4 N5 H5 N6 H6 N7 H7

β actin β actin

A

B

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.

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

24

Normoxia (control)0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

Rela

tive

dens

ity o

f ARN

T (f

old

chan

ge)

Hypoxia

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).

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).Normoxia

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Rel

ative

den

sity

of A

RNT

(fol

d ch

ange

)

Hypoxia

A

B

N1 H1 N2 H2 N3 H3 N4 H4 N5 H5 N6 H6 N7 H7

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 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).

25

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 βactin 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 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.

26

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 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

27

skeletal muscle 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 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).

28

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% 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%).

29

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 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

30

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 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 decreased SOCS3 protein

31

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.

Acknowledgements

32

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.

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