Consequences of Prenatal Hypoxia and the Development of Cardiovascular Disease

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Consequences of Prenatal Hypoxia and the Development of Cardiovascular Disease

Abstract:

Background: Cardiovascular disease is one of the leading causes of morbidity and mortality. Intrauterine programming is not fully understood but has an established role in developing a predisposition to cardiovascular disease following prenatal hypoxia. This predisposition is thought to relate to several factors: alterations in calcium handling during excitation-contraction coupling, mitochondrial dysfunction and the production of reactive oxygen species (ROS), amongst others. Initial research has identified an increase in respiratory capacity and decreased ROS production in female mice and a trend may indicate that the opposite occurs in males. Both the mechanism and the extent of these changes remain unknown.

Aims: The aims of this study were to establish whether changes in expression of Electron Transport Complexes I-V, NADPH Oxidase 4 (NOX4), Glutathione Peroxidase1 (GPx1)or SERCA-2A, were observed in the left ventricles of male and female mice following a 12-day prenatal hypoxic period compared to controls. Western blot analysis was performed on left ventricular samples of these mice.

Results: Results indicate that there may be a significant increase in subunit 1 of Complex IV in males following prenatal hypoxia when compared to controls. No other results were significant.

Conclusion: A rise in Complex IV expression in hypoxic males may indicate the presence of an underlying compensatory mechanism in order to increase ATP production and enhance oxygen uptake. Incomplete compensation causes suboptimal efficiency of the ETC and this has been shown to cause increased levels of ROS, which was observed in hypoxic males during preliminary research.

1.0 Introduction

While our understanding of the aetiology, pathophysiology and management of cardiovascular disease continues to develop, it remains a significant cause of morbidity and mortality1, 2,3. Since there is such a large patient population suffering from heart disease, there is a continued economic challenge to healthcare budgets. There are a number of well-studied risk factors associated with the development of cardiovascular disease, which include cigarette smoking, hypertension, obesity, diabetes mellitus and genetic inheritance, amongst others3, 4,5,6. More recent research has highlighted that other predisposing factors, including alteration to the prenatal environment, may increase the risk of cardiovascular disease in later life7. Evidence suggests that there is an increased chance of developing disorders across a spectrum of physiological systems as a result of foetal development in a suboptimal intrauterine environment. This includes subsequent impairment of the cardiovascular, metabolic, endocrine, nervous, reproductive, skeletal and respiratory systems7.

1.10 Intrauterine Programming

The mechanism in which changes to the intrauterine environment have a subsequent negative, non-reversible, impact on the development of tissues is referred to as intrauterine programming (IP)7. The concept of IP being involved in the construct and functionality of tissues following birth can be observed across a range of mammalian species, including rats and primates. Additionally, studies on the human population have identified an association between intrauterine growth restriction (IUGR) and the development of cardiovascular disease, and diseases to a number of other physiological systems.

The regulation of IP is influenced by a number of factors7. This includes prenatal nutritional supply, hormonal concentrations and oxygen levels. Reducing micronutrient or macronutrient supply, induced through calorific reduction, impairment of placental uptake or decreasing blood flow at the umbilical or uterine circulatory system, has been shown to have a negative impact on foetal growth, resulting in changes to postnatal physiology through impairment of cardiovascular, metabolic and endocrine systems.

In terms of endocrine influence of IP, the changes to factors such as nutrition and oxygen supply have been shown to cause changes to the concentrations of a wide range of hormones required for normal growth and development7. These changes to the prenatal environment have been shown to cause a reduction in foetal sex steroid hormones and increase foetal catabolic hormone concentrations. This has a subsequent effect on the development of the foetus through changes to utilisation of nutrients in both the foetus and the placenta7. Evidence indicates that alteration of glucocorticoids, sex steroids and thyroid hormone levels has a long-term detrimental effect on the cardiovascular, metabolic and reproductive systems. Studies on a number of different animals have shown an association between high levels of glucocorticoids and the development of hypertension, hyperglycaemia with insulin resistance, and a dysfunctional hypothalamic pituitary axis following birth7. The association between malnutrition and IP can be stopped if glucocorticoid production in the mother is inhibited, which suggests that the influence of glucocorticoids on IP may also be involved in the relationship between dysfunctional physiological systems associated with nutritional deficit during foetal development7.

1.11 The Influence of Oxygen on Intrauterine Programming

Oxygen supply to the foetus is essential for normal development. The induction of chronic intrauterine hypoxia is associated with the development of asymmetrical IUGR in rats and causes detriment to cardiovascular function in adulthood7. Additionally, high altitude induced hypoxia is associated with low birth weight and IUGR. It is however difficult to rule out the extent at which malnutrition may play a role in this trend, since communities living at high altitudes are often in areas where food and other resources are scarce7. Nevertheless, studies looking at hypoxia in chick embryos have identified a relationship solely between chronic hypoxia and IP of the cardiovascular system, which results in alterations in blood vessel and cardiovascular function7. It has been shown that the induction of hypoxia to chick embryos will result in increased sympathetic responses and impairment in the dilation response to nitric oxide in adult birds8. Changes to cardiac output distribution, to predominantly support the brain and the heart, and an increase blood pressure have also been observed in hypoxic chick embryos.  There is a similar trend seen in rats where hypoxia during foetal development causes a reduction in endothelium-dependent vasodilatation in the vascular supply to the abdominal viscera7.

The duration of oxygen depletion during foetal development has shown to be of importance with regard to the foetal physiological response8. In the acute setting, the foetal cardiovascular system responds to reduce peripheral vascular supply to ensure that there is optimal brain perfusion. This response is mediated by the carotid chemoreceptors and results in both the release of local and widespread chemical mediators of vasomotor tone, including epinephrine, norepinephrine and vasopressin8. There is additional release of local nitric oxide (NO) and reactive oxygen species (ROS) in response to hypoxia. Coupled with the release of the other factors, it is the ratio between NO and ROS that determines vascular tone in the developing foetus8.

Should hypoxia continue, the response to divert blood flow from the peripheral tissues will be maintained, which can lead to the development of disproportionate IUGR8. Increased arterial pressure with aortic hypertrophy and increased ejection pressure in the heart is also observed, resulting in elevated blood pressure, the development of arteriosclerosis and ischaemic heart disease. Chronic hypoxia also causes an increased sympathetic supply to peripheral circulation, which aids in the development of IUGR8. Aortic hypertrophy is of particular significance since it is a key predictor for the development of cardiovascular disease. Monitoring pulse-wave velocity of the aorta is a superior predictor for cardiovascular disease and impaired function of the left ventricle in comparison to monitoring of systolic blood pressure8.While the understanding of the influence of IP on changes to structure and function of the heart continues to develop, the underlying mechanisms responsible for these changes remain poorly understood10. Nevertheless, changes in metabolic function at the level of the mitochondria have been suggested to have a role in such changes10.

1.20 Mitochondrial Production of Reactive Oxygen Species

Mitochondria are the main producers of cellular ATP, and are a major contributors to the production of reactive ROS10. The aim of eventually developing treatments that could prevent alterations in cardiovascular physiology, and predisposition to cardiovascular disease, may require inhibition or dampening of mitochondrial ROS production and treatment to address changes in expression and functionality of the proteins responsible for the production of ROS10.

ROS are produced during the process of respiration11. One key process in which ROS are formed is through incomplete reduction of oxygen within the final stages of the mitochondrial electron transport chain. ROS production utilises up to 2% of oxygen that is consumed12. Usually this is balanced against antioxidant availability, which subsequently protects the intracellular apparatus from damage11. However, oxidative stress increases ROS production, which may result in injury to the cell if levels of antioxidant cannot compensate for this rise. Examples of commonly produced ROS include superoxide anion and hydrogen peroxide. While ROS are often described as a harmful by-product of cellular processes, there are established functions that ROS serve, which includes cell signalling and the regulation of genetic expression13. During acute oxidative stress, increased ROS production may also serve to enhance resilience against insult to the cell. It is often long-standing increased ROS production that causes functional impairment, damage and cellular death13.

1.21 Mitochondria: Overview

Mitochondria are organelles that span a diameter between 1 and 10µm14. They are involved not only in ATP production but also in a series of other important cellular functions, including apoptosis and cell signalling, as well as calcium, sodium and potassium regulation within the cell14. Some of the key factors that influence mitochondrial processes include ROS and calcium concentrations. Mitochondria are found in a wide range of cells and are generally observed at higher concentrations in the cells of tissues that require higher quantities of oxygen for respiration, such as cells within the nervous system, muscles, including the heart, where mitochondria make up almost one third of cellular volume13. Interestingly, mitochondria are double folded organelles, resulting in the presence of an inner and outer membrane, which permits normal mitochondrial function14. The outer membrane contains channels (porins) to allow free movement of small proteins across the outer membrane14. It additionally contains translocases, the active transporters of larger molecules through the outer membrane14.

The inner membrane, which is vital for ATP production and molecular exchange, has a resting membrane potential, which exists due to the respiratory function of mitochondria12. One key contributor to the presence of a membrane potential is the citric acid cycle (TCA). Pyruvate, the end product of glucose metabolism, is transported through the inner mitochondrial membrane where it is utilised within the TCA. The TCA produces NADH and FADH2, which are of particular importance because of their pivotal role to donate electrons (e) for utilisation within the electron transport chain, the chain in which ATP production occurs12, 15.

 

1.22 The Mitochondrial Electron Transport Chain

The main purpose of the Electron Transport Chain (ETC) is to produce energy in the form of ATP16. This is achieved through oxidative phosphorylation, occurring at the crista membrane, a fold within the inner membrane of mitochondria12, 16. Oxidative phosphorylation is the process in which there is cyclical oxidation of reduced coenzymes, reduction of oxygen to form water, and the production of ATP via the addition of phosphate groups (pi) to molecules of ADP12.

The ETC is made up of five key complexes, complex I to complex V16. Within the ETC, oxidation of NADH and FADH2  ultimately results in the release of ATP. NADH enters the ETC at complex I while FADH2 enters the ETC at complex II (figure 1). The ETC is driven by a proton gradient formed through movement of protons out of the inner mitochondrial matrix, into a space between the inner and outer membranes, known as the intermitochondrial space. This results in the formation and maintenance of a positive electrochemical gradient. Complexes I, III and IV are responsible for the generation of this electrochemical gradient through the movement of protons out of the inner mitochondrial membrane (figure 1). Energy, released from a decrease in membrane potential, permits the movement of protons across the inner mitochondrial membrane into the intermitochondrial space. This decrease in membrane potential arises from the movement of electrons across the ETC complexes15. Complex V, also referred to as ATP synthase, allows re-entry of protons back across the inner mitochondrial membrane, harnessing this potential energy to combine ADP and phosphate to produce ATP.  A small amount of protons may also leak back across the membrane without interaction with an ETC complex (figure 1)14.

There is an established role for the mitochondrial electron transport chain complexes as generators of ROS during sustained hypoxia, produced mainly at complexes I and III17.

Figure 1 – The Electron Transport Chain14.  I, II, III, IV represent their respective complex, ATP synthase represents complex V, ANT represents Adenosine nucleotide receptor and VDAC represents a voltage dependent anion channel of the outer membrane.

Promising results have shown that through the use of MitoQ, a mitochondrial antioxidant, a reduction inhypertension, decreased hypertrophy and enhanced endothelial function can be achieved in hypertensive rats10.This further supports the role of the mitochondrial ETC in both the production of ROS and the role in of ROS in producing a predisposition to cardiovascular disease in later life.

 

Preliminary research conducted at the Cardiovascular Research Department at the Core Technology Facility in Manchester has identified that there is a significant increase in the respiratory capacity of female mice that were exposed to prenatal hypoxia and a decrease in ROS production. There is also a trend to suggest that the opposite may occur in male mice that have experienced prenatal hypoxia. This poses an exciting challenge to identify whether there are true differing mechanisms between men and women in response to prenatal hypoxia, and to identify where any alterations in ROS concentration and respritatory capacity may be derived from. It remains unclear whether this could be as a result of alterations to the expression of components of the ETC, ETC functionality, changes to ROS producers external to the ETC, alterations to antioxidant availability or a combination of these factors. Completing the challenge of identifying where differences in the male and female response to prenatal hypoxia arise may provide a novel insight into the mechanisms responsible for the increased risk of developing cardiovascular disease in males. Understanding of such predisposition may subsequently result in the development of novel treatments to help reduce the risk of cardiovascular disease in later life.

1.30 Additional ROS Production

Additional ROS production occurs at the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzymes (NOX)18. Of the five NOX enzymes, NOX4 is of particular interest because of its relative abundant expression in cardiovascular tissue and its increased expression in a number of pathophysiological processes including hypertension, heart failure and non-haemorrhagic stroke.Another important difference of NOX4 in comparison to the other subtypes is its ability to produce ROS on a constant basis, without the requirement of external factors to influence such production. Research has highlighted the association between hypoxia in the prenatal stages of development and an increased expression of NOX4 in smooth muscle cells within the pulmonary vessels19. The mechanism responsible for this trend is through alterations to hypoxia inducible transcription factor 1α (HIF-1 α) expression, which increases in response to hypoxia.

1.40 Antioxidants

There are a number of antioxidants that are produced in order to combat the negative effects associated with ROS11. These are able to reduce ROS to less harmful products. Examples of key antioxidants include superoxide dismutase, responsible for the reduction of superoxide to hydrogen peroxide and glutathione peroxidase, which is capable of reducing hydrogen peroxide to water. This is of particular importance since hydrogen peroxide is able to escape from the mitochondria and access the cytoplasm, thus having the capability to cause intracellular damage and insults to DNA sequences.

1.50 Oxidative Stress & Cardiovascular Dysfunction 

Rat studies have identified an increased sensitivity to ischaemia/reperfusion (I/R) injuries in adults who experienced intrauterine hypoxia9.  This has been found to be more so in males who were fed a diet to promote obesity. This mechanism involves a decrease in the expression in protein kinase C epsilon (PKCε), which is known to be protective against ischaemia and reperfusion injury, following the induction of a hypoxic environment. It has additionally been shown that inhibition of PKCε in adults who had not experienced hypoxia during development would produce a similar sensitivity to I/R injury9. Noradrenaline is also able to induce a reduction in PKCε levels in hypoxic mice through the production of ROS. This shows a correlation between increased sympathetic innervation to cardiac tissue and a subsequent reduction in cardioprotective factors, such as PKCε, due to the production of ROS. Promising research has shown that ROS are able to modulate vascular tone, causing an increase in vascular resistance in the utero-placental circulation, resulting in impaired development and growth9. This is supported by studies that have reversed the reduction in the weight of newborn calves and mice through maternal supplementation with antioxidants9.

It has been suggested that long standing foetal hypoxia causes increased   oxidative stress within the cardiovascular system by the point of delivery9. In one particular study a pregnant mother was treated with vitamin C, an antioxidant. This prevented the maladaptive changes in the foetal cardiovascular system and was able to resolve both the increased cardiomyocyte contraction, caused by increased sympathetic supply, and the observed increase in peripheral resistance. This suggests that the provision of antioxidants during pregnancy may be able to correct cardiovascular dysfunction, highlighting the role of oxidative stress on IP of cardiovascular disease following prenatal hypoxia. Other studies have proven to be successful at preventing cardiac dysfunction following prenatal hypoxia through the use of other antioxidants. One study showed that N-acetyl-cysteine was able to prevent increased sensitivity to ischaemia/reperfusion injury in adulthood, through decreasing methylation of binding sites of PKCε gene, resulting in normalisation of PKCε levels. Treatment of heart samples that are sensitive to I/R injury with w-εRACK, a PKCε activator, results in enhanced recovery outcomes following I/R injury.

1.60 Additional Areas of Interest

In addition to the research that has highlighted the role of ROS and oxidative stress on IP of cardiovascular disease following prenatal hypoxia, there are other cellular changes that have been shown to cause a predisposition to cardiovascular disease in later life.

One potentially significant adaptation to the foetal cardiovascular system in response to prenatal hypoxia includes alterations to calcium handling and the subsequent effect on cardiac excitation-contraction coupling19.

1.61 Cardiac Excitation-Contraction Coupling

Cardiac excitation-contraction coupling is the mechanism responsible for control of cardiac contractility20. Calcium is vital for electrical signalling within the cardiovascular system and is required for contraction.

Calcium passes into the cell when voltage gated calcium channels open in response to depolarisation20. This subsequently causes a spike in intracellular calcium through activation of the sarcoplasmic reticulum. This rise in intracellular calcium results in conformational changes, allowing calcium to bind to troponin C, permitting cardiac contraction.

Following contraction, relaxation occurs when calcium is removed from the cell and troponin C is no longer stimulated20. This is achieved through a number of separate mechanisms, which involve the ATP-driven calcium channel at the sarcoplasmic reticulum, the sarcolemma, the Sodium /Calcium exchanger found at the sarcolemma, and the calcium uniporter channel found at the mitochondrial inner membrane20, 21.

A reduction in Sarcoplasmic reticulum Ca2+ ATPase (SERCA) protein expression, which is thought to be an adaptation to conserve energy at the expense of myocyte contractility, has been observed following prenatal hypoxia22. However, the extent to which SERCA levels change following a hypoxic intrauterine environment has varied between studies. Some studies indicate that there is a significant reduction in SERCA, where others have not shown any significant change in SERCA levels compared to controls22, 23.The alteration in SERCA levels following prenatal hypoxia is of particular significance due to the association between lowered SERCA expression and the development of heart failure24.

2.0 Aims

The aim of this study was to assess and compare the protein expression levels in cardiac tissues of male and female mice from normoxic and hypoxic pregnancies. Most studies have ignored sex-dependent differences, therefore this study may provide a novel insight into any changes observed between responses in males and females. SERCA-2A levels were measured and compared between 4 month old mice who had experienced either hypoxic or normoxic intrauterine conditions. Expression of NOX4 and the five mitochondrial electron transport complexes were measured. The final aim was to assess for any changes to Glutathione peroxidase 1 in order to identify any alterations to antioxidant concentration in response to prenatal hypoxia. All experiments were performed on the same group of mice.

3.0 Methods

Expression of SERCA-2A, NOX4, Electron Transport Complexes I-V and GPx1, were measured using western blot analysis of left ventricular tissue homogenates. Three repeats were performed for each experiment type excluding GPx, which was only performed once.

 

 

3.10 Sample preparation

Pregnant C57Bl/6J mice were used in all experiments. Mice were kept in a pathogen free environment in compliance with the standards set out in the Animals (Scientific Procedures) Act 1986 and experiments were conducted in adherence with the University of Manchester’s Animal Care Policies.

The environmental oxygen concentrations were controlled between day 6 and 18 of gestation. This was either at a ‘normal’ oxygen level (21%), or ‘hypoxic’ oxygen level (14%). Following oxygen control during this period, the mice were retained in a controlled environment that provided light exposure for 12 hours followed by 12 hours of darkness. Offspring were delivered into this environment and kept until 16 weeks, where they were then euthanised. Following cervical dislocation, hearts were removed and left ventricles were frozen in liquid nitrogen before being stored at -80°C. Analysis was performed on 7 male offspring (4 Normoxic, 3 Hypoxic) and 8 female offspring (5 Normoxic, 3 Hypoxic).

3.20 Protein Quantification

Samples were divided into 100mg sections before being further divided into 1mm pieces. These were then added to 1:100 RIPA lysis buffer containing 1% Nondiet p-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate and 1x PBS (NaCl 137mM NaCl, 10mM Phosphate, 2.7mM KCl, pH = 7.4). Protein inhibitors including 1:10000 Phenylmethylsulfonyl fluoride, 1mM sodium orthovanadate, 1:1000 aprotinin and 1:1000 leupeptin were added to the RIPA lysis buffer. Samples were subsequently homogenised for five seconds with ethanol, ddH20 and the RIPA lysis buffer. The homogeniser probe was washed between each homogenization to prevent contamination of samples. The homogenised samples were centrifuged at 14000Gfor ten minutes at 2 degrees celsius and were then quantified for protein content using Bradford microplate assay. 5µl of sample was added to 250µl of Bradford reagent and then incubated for 15 minutes at room temperature. The spectrophotometer was set to 595nm to measure the absorbance of light. Using Microsoft Excel, protein concentration was calculated from a standard curve derived from absorbance of light from the Bradford assay. Samples were stored at -80°C until use.

3.30 Western Blot Analysis

Immunofluorescence of SERCA-2A, NADPH Oxidase 4 (NOX4), Mitochondrial Electron Transport Complexes (ETC) I–V and GPx1 was measured via Western Blot Analysis. Left ventricular homogenates were taken from a -80°C freezer and stored on ice during preparation before being refrozen. Each homogenate was defrosted no more than 3 times before disposal. Bio-Rad all-blue stained ladder standard and RIPA 1:100 solution (1% Nondiet p-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate and 1x PBS (NaCl 137mM NaCl, 10mM Phosphate, 2.7mM KCl, pH = 7.4) and the protein inhibitors) were taken from -20°C and 4°C freezer and fridge respectively and stored on ice during use. NuPAGE® reducing agent was kept at 4°C until required and was then immediately returned for storage.

Each homogenate was decanted into labelled tubes before being mixed with 5µl of NuPAGE® LDS (lithium dodecyl sulfate) Sample Buffer,2µl of 500mM NuPAGE® Reducing agent (dithiothreitol), which was added last, and the remaining quantity of 1:100 RIPA lysis buffer (1% Nondiet p-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate and 1x PBS (NaCl 137mM NaCl, 10mM Phosphate, 2.7mM KCl, pH = 7.4) and the protein inhibitors) to make up 20µl. Following addition of NuPAGE® reducing agent, samples were immediately placed on a Grant QBD1 hot block for ten minutes. 20µl of each sample was then loaded to electrophoresis gels. 7µl of Bio-Rad all blue stained ladder was added to each gel, along with 20µl of a protein standard which was kept the same across all experiments. Using a Bio-Rad PowerPac 300 power supply, electrophoresis was performed in XCell SureLock™ Electrophoresis Cells. 500µl of NuPAGE® Antioxidantwas added before electrophoresis was performed in 1x MES (50mM 2-Morpholinoethanesulfonic acid, 50mM Trisaminomethane, 0.1% Sodium dodecyl sulphate, 1mM EDTA, pH 7.3) or 1x MOPS (50mM 3-N-morpholinopropanesulfonic acid, 50mM Trisaminomethane Base, 0.1% Sodium dodecyl sulphate, 1mM EDTA, pH 7.7) running buffer.

Transfer to a 0.45µm Amersham Protran support nitrocellulose membrane was performed with an Invitrogen X-cell II™ blot module, placed on ice, where 50µl of NuPAGE® antioxidant and 50ml of Tris-buffered saline (TBS) (50mM Tris base and 150mM NaCl) was added to the transfer kit. Total protein detection was performed with Bio-Rad REVERT total protein staining and detergent wash kit. Total protein staining was achieved by incubation with 3ml of the REVERT total protein stain for 5 minutes followed by two washes with the detergent for 30 seconds, both in 50ml falcon tubes. Thiswas imaged on a Licor BioSciences Odyssey CLx (700nm channel).

 

Membranes were then blocked in 5% Bio-Rad Blotting-Grade Blocker mixed with TBS (50mM Tris base and 150mM NaCl). This was followed by incubation with the primary and secondary antibody in 1%Bio-Rad Blotting-Grade Blocker mixed with TBS (50mM Tris base and 150mM NaCl). Secondary antibody incubation was performed in darkness. Between blocking and each antibody incubation period, membranes were washed in 10ml of TBST (50mM Tris 150mM NaCl, 0.1% Tween®) for 3 sets of 5 minutes in 50ml falcon tubes.

Imaging was performed with Licor BioSciences Odyssey CLx and immunofluorescence was measured on Licor Sciences Image Studio™ by machine preset average band quantification where possible. Background comparison was performed with a pixel width of 1 to 3 pixels.

Table 1a: Methodology Controls

Target Protein Sample mass (µg) Hot block temperature (Celsius) Electrophoresis Gel Electrophoresis time (minutes) Transfer Time (minutes) Protein band identification
Serca-2A 5 72 (3) NuPAGE® 4–12% bis-tris gel (10 wells) (3) 60 (3) 60 Licor REVERT Total Protein stain
NOX4 20 72 (3) NuPAGE® 4–12% bis-tris gel (10 wells) 60 (3) 75 Licor REVERT Total Protein stain
ETC I-V 10 72 (1)

72 (1)

50 (1)

NuPAGE® 4–12% bis-tris gel (10 wells) (1)

NuPAGE® 10% bis-tris gel (10 wells) (2)

60 (1)

70 (2)

75 Licor REVERT Total Protein stain
GPx 20 72 (1) NuPAGE® 4–12% bis-tris gel (10 wells) (3) 40 (1) 75 Licor REVERT Total Protein stain

Table 1b: Primary Antibody Storage and Incubation Concentrations

Target protein Primary Antibody Storage Temperature (Celsius) Primary Antibody Concentration Incubation Period
SERCA-2A Santa-Cruz sc-73022 (Lot #A0808) SERCA-2A mouse monoclonal 4 1:5000 60 minutes
NOX4 Abcam Anti-NADPH oxidase 4 antibody (ab133303) -20 1:1000 60 minutes
ETC I – V Abcam Total OXPHOS rodent WB Antibody Cocktail (ab110413) 4 1:500 (1)

1:1000 (2)

Overnight

Overnight

GPx Abcam Anti-Glutathione Peroxidase 1 (ab22704) -20 1:1000 Overnight

Table 1c: Secondary Antibody Storage and Incubation Concentrations

Target protein Secondary Antibody Storage Temperature

(Celsius)

Secondary Antibody Concentration Incubation Period (Minutes)
SERCA-2A Santa-Cruz Goat-anti mouse (Lot#C60405-09) 4 1:10000 60
NOX4 Santa-Cruz Goat-anti rabbit (Lot#C60607-11) 4 1: 10000 60
ETC I – V Santa-Cruz Goat-anti mouse (Lot#C60405-09) 4 1:10000 60
GPx Santa-Cruz Goat-anti rabbit (Lot#C60607-11) 4 1:10000 60

3.40 Statistical Analysis

Data on immunofluorescence was gathered from Licor Sciences Image Studio™ and exported into Microsoft Excel spread sheets for further analysis.

Data was subsequently analysed for each experiment followed by a collective analysis of the repeated experiments to assess for differences in mean fluorescence in the different groups. In addition to the comparison of mean values, the standard error was calculated and bar graphs were generated using Microsoft Excel. An unpaired t-test was performed to compare for significance between normoxic and hypoxic males and females. Following this analysis using Microsoft Excel, information was entered into IBM SPSS Statistics 22 where a 2 way Analysis of Variance (ANOVA) was performed.

4.0 Results

4.10 The Electron Transport Chain Complexes:

Image 1 – Western Blot Immunofluorescence of ETC Complexes identified at 20 (complex 1), 30 (complex 2), 34 (complex 4), 40 (complex 3) and 53kDa (complex 5)

4.11 Complex I, II and III

Regarding expression of complex I, II and III, there was no statistical significance between expression of any of these complexes in male or female mice when comparing expression in normoxic and hypoxic groups (figures 2,3 & 4).

There did appear to be a trend in which there was a decrease in complex I in males and an increase in complex I expression in females following prenatal hypoxia (Figure 2), although the opposite trend was observed in one of three studies (Figure 2). 2-Way Analysis of Variance (ANOVA) =.193.

With regard to Complex II, collective inspection identified a trend indicating that expression of complex II may be increased in females following prenatal hypoxia (Figure 3), which achieved a significant result in one of the three experiments. 2-Way ANOVA =.321.

When looking at Complex III expression collectively across experiments, there did not appear to be any identifiable trends in males or females (figure 4). 2-Way ANOVA =.897.

Mean Post Natal Complex I Expression in Male and Female Mice

Figure 2 – Mean expression of Complex I over 3 Experiments in male and female mice following intrauterine normoxic or hypoxic exposure. p male =.528437583,  p female =.20112

Mean Post Natal Complex II Expression in Male and Female Mice

Figure 3 – Mean expression of Complex II over 3 Experiments in male and female mice following intrauterine normoxic or hypoxic exposure. p male =.702717759,  p female =.102472039

Mean Post Natal Complex II Expression in Male and Female Mice

Figure 4 – Mean expression of Complex III over 3 Experiments in male and female mice following intrauterine normoxic or hypoxic exposure. p male =.991190556, p female =.840722859

4.20 Complex IV

In male mice that had experienced intrauterine hypoxia, there was a significantly increased expression of complex IV in comparison to males who had not experienced intrauterine hypoxia (p=.015951729) (Figure 5). It is important to note that one set of results was excluded due to a number of anomalous data points within the experiment, creating an unreliable collective data set. Rather than selectively choosing data to include, the entire experiment was excluded. There was no significant difference in expression of complex IV in females who had experienced intrauterine hypoxia in comparison to those who had not. Additionally, the combination of results did not identify any trend in changes to expression of complex IV in female mice. 2-Way ANOVA =.054.

 

 

 

Image 2 – Complex IV expression in normoxic and hypoxic females and males. Bands identified at 37kDa.

Collective Post Natal Complex IV Expression in Male and Female Mice

Figure 5 – Mean expression of Complex IV over 2 Experiments in male and female mice following intrauterine normoxic or hypoxic exposure p male =.015951729, p female =.764056699

4.30 Complex V

While each experiment found that there was, to some degree, a greater expression of Complex V in both male and female mice that had experienced prenatal hypoxia (Figure 6), the mean difference in expression of complex V had no statistical significance. 2-Way ANOVA =.619.

Collective Post Natal Complex V Expression in Male and Female Mice

Figure 6 – Mean expression of Complex V over 3 Experiments in male and female mice following intrauterine normoxic or hypoxic exposure. p male =.842931988, p female =.4303299

4.40 NADPH Oxidase 4 (NOX4):

NOX 4 expression was not significantly different in either male or female mice that had experienced prenatal hypoxia in comparison to those that had not. All three experiments (Figure 7) yielded similar results in which expression in NOX4 was similar across all groups. 2-Way ANOVA =.717.

  Hypoxic Female   Normoxic Female   Hypoxic Male      Normoxic Male

Image 3 – NOX4 expression in hypoxic and normoxic females and males. Bands identified at 67kDa.

 

Collective Post Natal NOX4 Expression in Male and Female Mice

 

Figure 7 – Mean expression of NOX4 over 3 Experiments in male and female mice following intrauterine normoxic or hypoxic exposure. p male =.743304967, p female =.418688425

4.50 Glutathione Peroxidase 1:

Due to time limitations, GPx1 expression was only measured once. It is also important to note that immunofluorescence imaging on the Licor Bioscience Odyssey CLx had a number of non-specific bands. Time restraints prevented repeat experiments and therefore optimisation of the western blotting protocol for this protein was not achieved. It was not possible to use a blocking peptide in order to be certain as to which band identified as GPx1. However, other laboratories had identified that the correct weight for GPx1 was 24kDa and subsequently the band at 24kDa was analysed25.

Hypoxic Male            Normoxic Male          Hypoxic Female        Normoxic Female

Image 4 – GPx1 expression in hypoxic and normoxic males and females. Bands identified at 24kDa.

There was no statistical significance to expression of glutathione peroxidase 1 in male and female mice when comparing expression in those that had experienced prenatal hypoxia with those that had not (figure 8). 2-Way ANOVA =.887.

Post Natal GPx1 Expression in Male and Female Mice

Figure 8 – Experiment 1 showing expression of GPX1 in male and female mice following intrauterine normoxic or hypoxic exposure. p male =.669104265, p female =.667322627

4.50 SERCA-2A:

SERCA-2A expression was not statistically different in groups exposed to prenatal hypoxia when compared to groups that had experienced a normoxic intrauterine environment. When looking across each experiment there is no apparent trend to note (Figure 9). 2-Way ANOVA =.815.

Image 5 – SERCA-2A expression in hypoxic and normoxic males and females. Bands identified at 60kDa.

 Hypoxic Male      Normoxic Male      Normoxic Female      Hypoxic Female

Collective Post Natal SERCA-2A Expression in Male and Female Mice

Figure 9 – Mean expression of SERCA-2A over 3 Experiments in male and female mice following intrauterine normoxic or hypoxic exposure. p male =.611905154, p female =.428335957

 

 

 

 

 

 

5.0 Discussion

This study has found that the only significant change to any of the electron transport chain complexes was in complex IV, cytochrome c oxidase (COX), where male mice that had experienced prenatal hypoxia had significantly increased expression. This may suggest that complex IV is up regulated in males in response to prenatal hypoxia. The role of Complex IV in regulating ETC efficiency has been identified in previous studies26. A response to increase Complex IV expression in hypoxic conditions could be a mechanism to increase ATP production and enhance the rate of oxygen uptake, which has been reported in studies looking at expression of subunit 4 of COX26. These results may not appear to provide much insight into why ROS have been seen to increase following prenatal hypoxia, since complex IV is capable of retaining all partially reduced products until they have been fully reduced, unlike complexes I & III27. However, a significant rise in COX does not necessarily indicate a complete compensatory response. Suboptimal ETC efficiency causes increased production of ROS through increased transfer of electrons to oxygen at complexes I and III26. Perhaps this is related to the increased ROS production seen in hypoxic males.

 

The primary antibody used in this study was specific for cytochrome c oxidase subunit I (COX-1). With regard to sub units 1, 2 and 3, chronic hypoxia in rats has been shown to increase the quantity of these specific sub units28. It has been suggested that this may be a compensatory mechanism in response to reduced respiratory function28. However, this was observed in adult rats and not offspring following maternal hypoxia. Additional studies on ewes have identified a rise in both COX-1 and COX-2 in foetal perirenal fat following maternal hypoxia when compared to controls that did not experience intrauterine hypoxia29. Both of these studies have additionally identified an increase in mitochondrial transcription factor-α (mtTFA), which is thought to regulate expression of cytochrome c oxidase28, 29. While these results highlight such a difference, it is not entirely clear whether this difference is in some way related to a response to increased ROS production, a decrease in ETC function, or a result of another pathological process.

Although not statistically significant, there did appear to be some trend towards an increase in expression of Complex I and II in females that had experienced prenatal hypoxia. There was also a trend in which complex I appeared to be down regulated in males. If this trend turned out be of significance in future studies, it may suggest an explanation for the increased respiratory capacity in females following hypoxia, and the reduction in male respiratory capacity, although it would not provide direct insight into why ROS were significantly lower in females.

NADPH oxidase 4 expression was not altered in hypoxic males or females. This indicates that NOX4 is not responsible for increased ROS production following prenatal hypoxia. While studies have observed an increase in NOX4 following intrauterine hypoxia, this has been seen in the smooth muscle cells of pulmonary vessels, which may respond to intrauterine hypoxia differently to the mitochondria of left ventricular homogenates.

While there was not a significant difference in GPx1 expression, it is challenging to make certain comments on GPx1 expression following prenatal hypoxia, since the experiment was only conducted once. Before coming to any conclusions regarding the expression of GPx1 following prenatal hypoxia, more work would need to be performed on this antioxidant. It would also be reasonable to perform an experiment with the use of a blocking peptide to ensure that the correct band was analysed. Additional work to optimise procedures may be able to reduce non-specific binding in the future.

Regarding SERCA-2A expression, there was no significant difference between expression of SERCA-2A following prenatal hypoxia when compared to control groups. This result is supported by research which has indicated no change to SERCA-2A levels in left ventricular samples23. Similar to the suggestions regarding NOX4 expression, different cell-types appear to respond differently to prenatal hypoxia.

5.10 Methodology Strengths and Limitations

Some of the key strengths of this study include the repetition of each experiment. Repeating each experiment 3 times, except for GPx1, allowed anomalous results to be identifiable, and ensured that results can be repeated in future experiments. Additionally, in NOX4, ETC I-V and GPx1 analysis, samples were loaded ensuring that each group (male, female, normoxic and hypoxic) was distributed across both gels in an attempt to prevent transfer issues from impacting results. All experiments were conducted with the same equipment. Both primary and secondary antibody brands were also kept the same, and antibody incubation conditions were kept the same within each experimental set. In order for protein degradation to be minimised, it was ensured that samples were kept on ice during use and defrosted no more than 3 times before disposal. All chemicals were also stored in the appropriate conditions. These consistencies add to the validity of the results produced.

Limitations to this study include the changes between experiments looking for the electron transport chain proteins. This included a change in temperature of the hot block for one experiment, to identify if there was any significant change in result. This was because it was thought that 72°C might have been high enough to cause damage to some of the ETC complexes, which could have affected the results. Another alteration between experiments includes the primary antibody concentration. A concentration of 1:500 was used in one experiment, and a concentration of 1:1000 was used in the other two. It is however worth noting that the extent of these changes are limited since all samples were standardised against their own protein standard, within each experiment, before data was analysed collectively. In addition, the electrophoresis gels were changed from 4-12% Bis-Tris to 10% Bis-Tris Gels, in order to allow for better separation of fluorescent bands. GPx1 western blotting was only performed on one occasion due to time constraints. Consequently it is more challenging to draw conclusions from the results and more work must be done on GPx1, in particular, before definitive conclusions could be made.

5.20 Conclusion

In summary, complex IV expression may be upregulated in response to prenatal hypoxia in males. Specifically, subunit 1 expression appears to be increased, which may be due to increases in mitochondrial transcription factor α (mtTFA). This indicates a degree of mitochondrial involvement in the response to low levels of oxygen in the intrauterine environment. Perhaps a suboptimal ETC function existing beyond increased complex IV expression in males is responsible for the observed increase in ROS production.

It is clear that more research needs be performed to evaluate changes to the cardiovascular system following prenatal hypoxia. Work focussing to highlight the true nature of ROS response and respiratory capacity changes following hypoxia would be beneficial and would set the foundation for further study. With regard to the proteins investigated in this study, repeated experiments on larger sample groups would be beneficial. It would be interesting to investigate changes to other subunits of Complex IV, and subunits of the other ETC complexes, following prenatal hypoxia. This may provide further insight into the functional changes that occur following oxygen restriction in the intrauterine environment. It would be of benefit to optimise methods for each protein initially and ensure that all controls are kept the same within each experiment. In addition, more work looking firstly at glutathione peroxidase 1, followed by other antioxidants, may assist in furthering our understanding of antioxidant responses following prenatal hypoxia. Again, it would be useful to continue to separate the evaluation of changes to protein expression between males and females as this may provide useful information regarding factors that could put men at a greater risk of cardiovascular disease, and conversely, why cardiovascular disease is less common in women.

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