Department of Physics, Chemistry and Biology
Final Thesis
The effects of prenatal hypoxia on the levels of
the Gsα and Giα protein subunits in the heart of
the Broiler chicken (Gallus gallus)
Nabil Rashdan
Supervisor: Jordi Altimiras, Linköpings universitet
Examiner: Cornelia Spetea Wiklund, Linköpings universitet
Department of Physics, Chemistry and Biology
Linköpings universitet
SE-581 83 Linköping, Sweden
Content
1 Abstract………………………………………………………………………………….........
2 List of abbreviations……………………………………………………………………..........
3 Introduction………………………………………………………………………………..….
4 Material and Methods…………………………………………………………………....……
4.1 Incubation conditions…………………………………………………………………..……
4.2 Handling of chickens…………………………………………………………..........
4.3 Sample preparation…………………………………………………………...……..
4.4 Gel electrophoresis………………………………………………………………......
4.5 Western blotting………………………………………………………………..........
4.6 Antibodies……………………………………………………………………...……
4.7 Statistical analysis………………………………………………………………...…
5 Results…………………………………………………………………………………...…….
5.1 Effects hypoxia on growth………………………………………………………..…
5.2 subunits in the embryo………………………………………………………………
5.3 subunits in the Juvenile ……………………………………………………….…….
6 Discussion……………………………………………………………………………………..
6.1 Effects of prenatal hypoxia on the Gα subunit…………………………………...…
6.2 Effects of prenatal hypoxia downstream of the Gα subunit………………….……...
6.3The protective role of Giα in the developing heart…………………………………..
7 Acknowledgements……………………………………………………………………………
8 References……………………………………………………………………………………..
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X Introduction
The process by which an organism grows and develops has a great impact on the future outcome
of the individual, affecting its survivability in the environment it eventually emerges into and its
success to procreate. Two factors affecting these processes or developmental pathways are the
organisms genetics, inherited from its parent(s), and the environment in which this development
occurs. Most vertebrates are sensitive to environmental stresses, which allow them to take altered
developmental trajectories in an attempt to reach a suitable phenotype for that environment. This
sensitivity may sometimes have adverse effects that resonate into maturity and possibly into
following generations. In humans chronic diseases such as cardiovascular disease and diabetes
have been associated with low birth weight due to reduced maternal nutrition (Barker, 2001).
Environmental factors that affect development include, but are not restricted to, incubation
temperature, fetal nutrition, and the availability of oxygen.
The reduced availability of oxygen, hypoxia, is an environmental stress that could be brought
about in clinical conditions such as placental insufficiency, cord compression, preeclampsia, and
anemia. An increase in fetal heart to body weight ratio was found in rats exposed to hypoxia
compared to normoxic controls (Li et. al . 2003, Netuka et. al . 2006), furthermore the hearts of
rats exposed to prenatal hypoxia were also less tolerable than the normoxic controls to ischemic
insult. In sheep hypoxia due to fetal anemia causes an increase in cardiac output and similar to
rats an increase in fetal heart to body ratio (Davis et al 1999). Broiler chickens exposed to
chronic fetal hypoxia showed decreased body mass, reduced ventricle wall mass (Lindgren &
Altimiras, 2009), cardiomyopathy, and diastolic dysfunction (Tintu et al. 2009). The sensitivity
of beta adrenergic receptors in these chickens was found to increase in the embryonic stages but
decreased postnataly (Lindgren & Altimiras, 2009).
A potential explanation for these differences in sensitivity is a shift in β 2-adrenergic receptors
from a Giα to a Gsα signaling pathway in the embryonic stages and vice versa in the postnatal
stages. β2-Adrenergic receptors have been shown to play a role in regulating cardiomyocyte
survival, where they have been associated with both pro- and anti-apoptotic effects, this dual
signaling effect has been found to be the result of a Gsα/ Giα shift (Zhu et al. 2001) that occurs
when stimulation of the Gsα -coupled receptors leads to an increase in intracellular cAMP. In
turn, cAMP causes increased cAMP-dependent protein kinase (PKA) activation. PKA-mediated
phosphorylation of the β2-adrenergic receptors decreases its affinity for Gsα and increases its
affinity for Giα and subsequent activation of the β2-Adrenergic receptors leads to increased Giα
signaling (Daaka et al. 1997). Gsα/ Giα shifting was further demonstrated in transgenic mice with
cardiac specific over expression of β2-adrenergic receptors which resulted in both a positive and
a negative inotropic response to isoprenaline (Hasseldine et. al. 2003). Furthermore a study by
Martin et. al. (2004) found strong evidence in CHO (Chinese Hamster Ovary) cell cultures that
β1-adrenergic receptors also undergo a PKA-mediated Gsα/ Giα shift. this study attempts to
investigate this explanation by measuring the relative concentration of both G protein alpha i & s
between animals incubated in chronic hypoxia and normoixic a normoxic control.
Maternal responses would have reduced the effects of hypoxia on the embryo, further more
catecholamines and other hormones readily cross the placenta, these confounding effects were
avoided by using a egg-developing animal model. A fast growing strain of Broiler chicken was
used in this study that has been shown to respond to hypoxia in a similiar fashion to mammalian
models, and has been used in a previous study by this group (Lindgren & Altimiras, 2009),
additionally this study attempts to explain some of the results obtain in the previous study,
making
the
use
of
the
same
model
appropriate.
X Materials and Methods
X.1 Incubation conditions and sampling
The fast-growing strain Ross 308 of the broiler chicken (Gallus gallus domesticus) was used for
this study. The eggs were obtained from a local hatchery (Kläckeribolaget, Väderstad, Sweden).
The eggs were stored at a constant temperature of 18 ˚C and turned twice daily until incubation
(Hova-bator, Invansys . Periodic candling of the eggs was preformed to monitor fetal
development and viability. Prior to incubation the eggs were weighed to the nearest hundredth of
a gram.
The eggs were incubated at 37.8 ºC with a relative humidity of 45% and turned once every hour
(model 25 HS, Masalles Comercial, Barcelona, Spain). The normoxic control group (N) was
provided with ambient air where as the hypoxic group (H) was supplied with a mixture of
nitrogen and air using standard rota-meters (B-125-50, Porter Instruments company, hatfield,
Pennsylvania, USA) achieving a final isobaric oxygen concentration of 14%, which was chosen
based on previous studies (Lindgren and Altimiras, 2009) which found that oxygen
concentrations of 12% resulted in a mortality rate greater than 75% before developmental day 5.
The oxygen concentration was monitored every ten seconds using a galvanic oxygen sensor
(Pico Technology Inc, Cambridgeshire, UK). On day 20 of incubation the eggs were placed in
hatching trays in the bottom of the incubator resulting in the chickens being hatched in the same
oxygen concentration they were incubated in.
Hatchlings were weighed to the nearest tenth of a gram and released into pens with free access to
commercial chicken feed (Allfoder PK, Foderfabrik, Västerås, Sweden) and drinking water.
A light intensity of 6 lux at floor level was maintained with a 12h light/dark photo-period. The
general room temperature was set to 25 °C while the temperature was kept between 30 and 32 °C
for the first week and 28 to 30 °C for the remainder of the period. The animals were weighed on
weekly bases to monitor growth.
35 Day post hatching juvenile chickens and 19 day old embryos were used for experiments based
on previous studies (Lindgren and Altimiras, 2009), referred to as P35 and E19 respectively. At
these times the animals were euthanized by decapitation and weighed (Sartorius BP 221S,
Sartorius, Goettingen, Germany). The embryonic mass did not include the yolk sac. The animals
were immediately dissected and the hearts excised and rinsed in modified Ringer's buffer
solution (138 mM NaCl, 3 mM KCl, 3 mM CaCl2, 1.8 MgCl2, 10 mM HEPES, Tris to pH 7.4).
The hearts were blotted and weighed to the nearest milligram. The dissected ventricles (P35) or
the whole heart (E19) were flash frozen in liquid nitrogen and stored at -80 °C until further
processing.
All procedures were approved by the local Ethical Committee (Linköping, Sweden)) diary
number 48-04.
X.2 Sample preparation and analysis
Samples were thawed on ice then homogenized using a electric homogenizer (Ultra-Turrax T8,
IKA® Werke GmbH & Co. KG, Staufen, Germany) at medium speed for 30 seconds in 1 ml
RIPA buffer (Tris.HCl 50mM, NaCl 150 mM, SDS 0.1 %, Sodium Deoxycholate 0.5 %, Triton
X 100 1, pH 7.4 ) to which 10 µl of Halt Protease Inhibitor Cocktail with 0.5mM EDTA (Pierce
Biotechnology, Rockford, Illinois, USA) was added. Roughly 80mg of left ventricle tissue was
used for the juveniles samples whereas the whole hearts were used for the embryos. The samples
were then placed on a shaker (PolyMax 2040, Heidolph Elektro GmbH & Co. KG, Kelheim,
Germany) on ice at 15 rpm for 30 minutes, and centrifuged for 10 minutes at 14,000 rpm
(MiniSpin plus, Eppendorf AG, Hamburg, Germany), the supernatant was divided into 200 µl
aliquots and frozen at -80 until further processing. Total Protein concentration was determined
using bicinchoninic acid assay microplate assay per manufacturers’ instructions (Pierce
Biotechnology, Rockford, Illinois, USA).
X.3 SDS-PAGE
After the BCA assay the protein samples were diluted to 25μg/10μl, this concentration was
selected after a series of dilutions were tested and was found to yield the highest signal without
saturation. the sample was then mixed with loading buffer (Tris 250 mM, SDS 0.4%, Glycerol
46%, Dithiothreitol 120 mM, Bromophenol blue 0.4%) at a ratio 2:1 (Sample : Loading buffer)
the mixture was then heated to 45°C for 7 minutes and left to stand at room temperature for >30
minutes. 15 μl of the treated sample was separated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE), the stacking and separating gel contained 6% and 14% acrylamide
respectively, both contained 36% Urea. A juvenile brain homogenate known to be rich in both
Gα-subunits was used as an internal standard using a dilution of 25μg/10μl.
X.4 Western blot
After electrophoresis the proteins were semi-dry electroblotted (2117 electrophore II, LKB
instruments, Mt Waverley, Victoria, Australia) onto polyvinylidene difluoride (PVDF)
membranes (GE healthcare Piscataway, New Jersey, USA). The gels were then stained using
PageBlue Protein stain according to manufacturers’ instructions (Fermentas, Burlington, Ontario,
Canada) to check for transfer efficiency. The membranes were washed in TBS buffer (Tris.HCl
50mM, NaCl 100 mM, pH 7.4) for 5 minutes, then blocked in 5% non-fat milk in TBS buffer for
1 hour, after which the membranes were washed with TTBS buffer (Tris.HCl 50mM, NaCl 100
mM, 0.1% Tween 20, pH 7.4) for five minutes, followed by overnight incubation with a 1:10000
dilution of primary antibody (anti-Gsα or Giα) in TTBS buffer with 0.2% Amersham ECL
Blocking Agent (GE healthcare Piscataway, New Jersey, USA) at 4°C. The membranes were
then washed 3 times with TTBS buffer for 5 minutes, followed by a 1 hour incubation with a
1:3000 dilution of secondary antibody in TTBS with 0.2% Amersham ECL Blocking Agent (GE
healthcare Piscataway, New Jersey, USA) for 1 hour at room temperature. The membranes were
washed 3 times with TTBS buffer for 5 minutes, followed by two 5 minute washes with TBS
buffer. The membranes were developed by incubating them with 2 ml Amersham ECL Plus
Western Blot Detection System (GE healthcare Piscataway, New Jersey, USA) for 5 minutes and
visualized using a CCD camera (LAS-4000mini, Fujifilm, Minato, Tokyo, Japan). After
exposure for 3 and 5 minutes, the blots were analyzed using Fujifilm Multi Gauge software v3.2.
X.5 Antibodies
The rabbit anti- Gsα antibody and the rabbit anti- Giα 1 & Giα 2 antibody were purchased from
Calbiochem Inc., where as the secondary antibody ECL Donkey Anti rabbit IgG, peroxidase
linked whole antibody was purchased from GE healthcare.
X.6 Statistical analysis
Values are expressed as a percentage of the internal standard, and the mean was used as a single
entity for statistical analysis. Paired Student’s t-test was used to determine the difference
between control and treatment groups.
X Results.
X.1 Hypoxia impaired embryonic growth and increased relative heart mass
The body mass of embryos incubated in hypoxia was significantly lower (17%) than that of the
normoxic control, this difference in body mass disappeared with age as no significant difference
was observed in the juveniles (Table 1), although, relative heart mass was significantly increased
in the hypoxic embryos by 7% and persisted in the juveniles, indicating cardiac enlargement
(Table 1).
E19
Normoxia
Hypoxia
P35
Normoxia
Hypoxia
Body Mass, g
Heart to body
Mass Index
Protein μg/mg
32.54 (2.63)
27.03 (2.78)*
0.589 (0.06)
0.628 (0.073)*
71.95 (22.13)
77.63 (18.56)
993.17 (253.73) 0.446 (0.050)
1011.24 (225.01) 0.480 (0.060)*
85.40 (25.72)
99.75 (25.00)
X.2 The chicken heart showed differential Gsα isofroms
The anti-Gsα antibody used in this study is capable of binding to all Gsα isoforms , Gsα -small
(45 kDa) and Gsα-large (52 kDa) and their splice variants (Mumby et al. 1991). Both isoforms
were detected in the internal standard, but the predominant immunedetectable isoform was the 45
kDa Gsα -small isoform (Fig. 1)
X.3 Hypoxia increased Giα in the embryo
Hypoxia had no effect on the level of Gsα in the E19 group, no significant difference was found
between the hypoxic and the control group, while the level of Giα was significantly more in the
hypoxic group than in the normoxic control (115% and 94% of the internal standard
respectively).
X.4 Hypoxia caused an increase in Gsα in the juvenile
In contrast to the embryos, hypoxia significantly increased Gsα in the juveniles by 21,5%
compared to the normoxic control, though no significant difference in Giα levels was found
between the hypoxic and normoxic groups.
X Discussion
Chronic prenatal hypoxia increases the sensitivity of β-ARs in the embryonic heart, while
decreasing the sensitivity of these receptors 5 weeks post hatching (Lindgren and Altimiras,
2009). Unexpectedly the relative amount of Gsα in the embryonic chicken was found to be
unaffected by chronic prenatal hypoxia while Giα increased. In contrast to the embryos, the
hypoxic juvenile samples showed no significant change in the relative amount of Giα. While a
significant increase in Gsα was detected which agrees with previous studies (Hrbasová et al.
2003, Pei et al. 2000), interestingly these studies found that hypoxia increases Gsα-large. The
effects hypoxia observed in this study were predominantly on Gsα -small. It is noteworthy to
mention that these studies investigate the affects of hypoxia on adult animals. An increase in
Gsα-Small only, is reported in a study investigating the effects of prenatal hypoxia on the adult
rat (Li et al. 2003). Although Gs-large is detected, no significant changes are observed between
treatments. The physiological importance of Gsα -small/ Gsα -large is not clear.
The effect of hypoxia on the functional activity of Gα subunits is a potential explanation for this
apparent dissociation between the levels of Gα subunits and the sensitivity of the β-ARs. As this
altered activity of the Gα subunits would result in changes in the sensitivity of the β-ARs
independent of the amount of Gα available. Several studies that found Gsα levels to be elevated
by hypoxia also found that hypoxia reduces the functional activity of this subunit (Hrbasová et
al. 2003, Kacimi et al. 1995, Pei et al. 2000) additionally, Feldman et al. (1991) found that Giα
activity is increase while Giα levels remain unchanged in dilated cardiomyopathy.
X.1 Prenatal hypoxia could affect the sensitivity of β-AR signaling downstream of the Gα
subunit
After stimulation of the β-AR it acts as a guanine nucleotide exchange factor, activating the Gα
subunit which in turn interacts with adenylyl cyclase, It has been suggested by Alousi et al.
(1991) that Gsα is in stiochiometric excess in the cell, and that adenylyl cyclase is the rate
limiting factor in β-AR signal transduction. This implies that if hypoxia affects adenylyl cyclase
activity, this effect would be more profound on the sensitivity of β-AR signaling. Indeed, studies
investigating the effects of hypoxia on cardiac β-AR found a marked decrease in adenylyl
cyclase activity (Hrbasová et al. 2003, Kacimi et al. 1995, Pei et al. 2000) one of these studies
found that hypoxia inducesd decrease in adenylyl cyclase activity persists after five weeks of
recovery (Hrbasová et al. 2003).
Rohlicek et. al. (2005) in a paper investigating the effects of neonatal hypoxia on the adult heart
of the rat suggest that hypoxia may induce a shift in adenylyl cyclase isoforms favouring a more
active isoform. Similar to the mammalian heart the adenylyl cyclase isoforms expressed in the
chicken heart are Adenylyl cyclase type 5 and adenylyl cyclase 6 (Yu et al. 1995) although these
isoforms exhibit similar levels of activity (Katsushika et al. 1992), experiments using adenylyl
cyclase 5 knockout mice found that the inhibitory effect of the muscarinic receptors on beta
adrenergic signaling mediated through Giα is insignificant compared to a 20% decrease in
contractile response in the wild type, these results imply that adenylyl cyclase is the primary
target of the inhibitory actions of Giα. (Okumura et al. 2003). Furthermore, neonatal cardiac
myocytes overexpressing adenylyl cyclase 6 show an increase in Akt protein activity which
protects the cell from apoptosis, this suggests increased adenylyl cyclase 6 may play a protective
role in the heart. (Gao et al. 2008). In contrast, adenylyl cyclase 5 inhibition prevents cardiac
myocyte apoptosis induced by β-AR overstimulation (Iwatsubo et al. 2004). During the normal
development of the rat heart the levels of adenylyl cyclase isoform expression changes inversely,
adenylyl cyclase 6 expression decreases with age (Tobise et al. 1994), while adenylyl cyclase 5
expression increases as the animal develops (Espinasse et al. 1995). It would be interesting and
informative to elucidate the underlying mechanisms of this change in expression ratio, and
whether or not hypoxia has an effect on it.
X.2 Giα plays a protective rather than inhibitory role in the developing heart
This study demonstrated that in the chicken model prenatal hypoxia increased the amount
of Giα in the heart of the embryo, despite an increase in β-AR sensitivity to epinephrine
previously reported in these hypoxic embryos (Lindgren and Altimiras, 2009). In the adult heart
inhibition of the Giα subunit with Pertussis toxin, a potent Giα inhibitor, significantly increases
the contractile response to the β2-AR selective agonist zinterol (Xiao et al. 1995, Xiao et al.
1999). Suggesting that Giα partially negates the Gsα mediated contractile response due to β2-AR
stimulation. Interestingly experiments investigating the inhibitory effect of Giα on β2-AR
signaling in the neonate, found that unlike the adult, in the developing heart Giα has little to no
inhibitory effect on the β2-AR (Aprigliano et al. 1997, Rybin et al 2003). Rybin et al. used
Pertussis toxin to block the effects of Giα in neonatal rat ventricular myocytes, and found no
significant difference between the Pertussis treated cells and the controls. Furthermore, the
contractile response of neonatal rat myocytes after β2-AR selective stimulation is comparable to
Pertussis toxin treated adult rat myocytes.
The β2-AR is capable of delivering an antiapoptopic signal that promotes cell survival (Zhu et al.
2001), that is mediated by Giα which activates the Gβγ subunits, which in turn recruit
phosphoinositide 3-kinase (PI3K) to the plasma membrane from the cytosol, PI3-K then converts
Phosphatidylinositol 4,5-bisphosphate (PIP2) to Phosphatidylinositol (3,4,5)-trisphosphate (PIP3),
PIP3 binds to Akt protein which results in growth factor mediated cell survival. (Chesley et el.
2000) demonstrated that hypoxia induces apoptosis in rat neonatal cardiac myocytes, and more
importantly pretreatment of these cells with zinterol protected them from hypoxia induced
apoptosis via the afore mentioned signaling pathway. The increase in Giα reported here could
possibly be a result of a similar protective mechanism in response to the hypoxic insult. This
increase in Giα cannot be entirely attributed to β2-ARs, other receptors such the M2 muscarinic
acetylcholine receptor also signal through Giα. And could contribute to the increase found,
although Chesley et al. (2000) also showed that stimulation of the M2 muscarinic acetylcholine
receptor with carbachol, a non selective cholinergic agonist, induced cell survival, suggesting
that the Giα-PI3K-Akt signal pathway is shared by the two receptors.
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