Download The Effect of Dobutamine on the 8

The Effect of Dobutamine
on the 8-Day Old Chicken
Embryonic Heart Rate
Amanda Lindsay
Fatema Kermalli
Biology 142 Section 002
Purpose
To evaluate the effect of different concentrations of
dobutamine on the 8-day old chicken heart rate.
Hypothesis
In this experiment it was hypothesized that
adding dobutamine to the 8-day chicken heart
embryo in vitro would increase the heart rate
(bpm) in relation to the concentration of the
drug (1.25 x 10-4 mg/ml to 1.25 x 10-2 mg/ml).
Chicken Heart Development
Occurs on the ventral surface from the fusion of paired
precardiac mesodermal tubes lying on either side of developing
foregut.
At 25-30 hours incubation:
Paired heart vesicles fuse
posteriorly to form a
continuous tube. The heart
tube, now ventral to the
foregut, has four distinct
regions through which blood
flows anteriorly.
McLaughlin and McCain, 1998
Heart
Bulge
http://embryology.med.unsw.edu.au/wwwhuman/Stages/stage11.htm
Chicken Heart Development
Heart regions:
– Conotruncus
– Ventricle
– Atrium
– Sinus Venosus
At 33 hours incubation:
Heart tube bends to form
an “S” shape.
At 48 hours incubation:
Heart forms a single loop
by folding upon itself.
Figure 1: Dorsal view of a
chicken embryo at 33 hours
incubation.
Figure 2: Right side of a
chicken embryo at 48 hours
incubation.
http://www2.lv.psu.edu/jxm57/chicklab/outline.html
McLaughlin and McCain, 1998
Chicken Heart Development
Figure 3: Right side of a chicken embryo
at 72 hours incubation.
http://www2.lv.psu.edu/jxm57/chicklab/outline.html
Heartbeat begins just after the
paired heart rudiments start to
fuse.
– After the fusion is complete,
the sinus venosus becomes the
embryonic pacemaker.
When the atrium and ventricle
divide, the sinus venosus gives
rise to the sinoatrial node, the
mature pacemaker, in the right
atrium.
McLaughlin and McCain, 1998
Vertebrate Heart Development
Derived from
the
embryonic
conotruncus
Derived
from the
embryonic
sinus
venous
Figure 4: Diagrammatic representation of the
anterior view of a mammalian heart.
http://www.clevelandclinic.org/heartcenter/images/guide/heartworks/insideheart3.jpg
Dobutamine
Synthetic drug approved for clinical use in 1978.
Must be continually administered intravenously
as the plasma half-life is only two minutes.
Catecholamine that directly works to increase
myocardial contractility without greatly
increasing heart rate.
Useful in treating acute cardiac failure with low
output.
Sonnenblick, 1979
Dobutamine
Isoproterenol
http://www.ch.ic.ac.uk/local/projects/j_hettich/salbutamol/images/dev2.gif
http://www.chem.ox.ac.uk/it_l
ectures/chemistry/mom/Dobu
tamine/Dobutamine.jpg
Dobutamine
Stimulates beta-1 adrenergic receptors located on the plasma
membranes.
This activates the Gs protein which releases the alpha
subunit.
The subunit activates
adenylate cyclase which
catalyzes the reaction converting
ATP to cAMP.
cAMP activates protein kinase A
which phosphorolates a protein.
This changes the protein’s
activity and causes a cellular
response.
Germann, 2005
http://kph12.myweb.uga.edu/11_12cAMP.jpg
Dobutamine
Affects of protein kinases increasing contractility
1. Greater flow of calcium allowed into the cell during an
action potential due to the phosphorylation of calcium
channels in the plasma membrane.
2. Enhanced calcium release due to the phosphorylation of
proteins in the sarcoplasmic reticulum (Germann, 2005).
• Beta-2 and Alpha receptors are stimulated slightly.
Table 1: Adrenergic-Receptor Activity of Sympathomimetic Amines
(Sonnenblick, 1979).
a
b1
b2
PERIPHERAL
CARDIAC
PERIPHERAL
NOREPINEPHRINE
++++
++++
0
EPINEPHRINE
++++
++++
++
DOPAMINE
++++
++++
++
ISOPROTERENOL
0
++++
++++
DOBUTAMINE
+
++++
++
++++
0
0
METHOXAMINE
Clinical Significance
Chicken heart development is similar to that of the human
embryo. As a result, the chick embryo serves as an excellent
model system to study the effects of cardiovascular drugs on
the developing human heart.
Developmental Stages of the Chick
http://www.microscopy-uk.org.uk/mag/imgnov04macro/series.jpg
Methods
Step 1: Serial Dilutions of Dobutamine
–
A 12.5 mg/ml aliquot of drug
was diluted 100 fold with media
CMRL to yield a 0.125 mg/ml
stock solution.
–
Serial dilutions of the stock
were created to obtain 1.25 x 104 mg/ml dobutamine, 1.25 x 103 mg/ml dobutamine, and 1.25
x 10-2 mg/ml dobutamine.
http://www.blcleathertech.com/images/
pagefiles/Corbis_test%20tubes.jpg
Methods
Step 2: Eggs were “windowed” according to the methods of
Cruz, 1993.
–
Scotch Magic tape was placed along the horizontal axis at
the top of the egg.
–
A hole was carefully punctured near the pointed end of the
egg and 1-2 ml of albumen were withdrawn using a 20 G
needle.
–
An oval opening was cut out, pulling up with scissors in
order to protect the embryo and vitelline envelope.
–
In vivo HR (bpm) was determined and recorded.
Methods
Step 3: The embryo was “explanted” according to the methods of
Cruz, 1993.
–
A Syracuse dish was filled with about a quarter of an inch of
warm chick media CMRL.
–
A filter paper “doughnut” was framed around the embryo and
then picked up. (The embryo spoon was used to lift out the
embryo if the “doughnut” technique failed.) Microdissecting
scissors were used to cut the embryo free of the egg
–
In vitro HR (bpm) determined and recorded.
Methods
Step 4: Application of Dobutamine.
–
As much media CMRL as possible was removed without
allowing the embryo to dry. The embryo was covered with the
1.25 x 10-4 mg/ml dobutamine. In vitro HR (bpm) was
determined and recorded.
–
Step one was repeated, using the 1.25 x 10-3 mg/ml
dobutamine dilution.
–
Step one was repeated, using the 1.25 x 10-2 mg/ml dilution
of dobutamine.
Methods
Repeated steps II, III, and IV on at least 6 embryos in order to
obtain enough data for quantitative analysis.
Explanation of the heart from the embryo
–Explanted the heart; some immediately after removal from the
egg and others following the embryo’s exposure to the highest
concentration of dobutamine (1.25 x 10-2 mg/ml). Obtained
the in vitro HR (bpm), and noted any arrhythmias.
Results
Heart Rate (bpm)
250
200
150
100
50
0
150 162 167 153 137
135 156 111 114 80
150 232 110 139 118
In Vivo
In Vitro
0.000125mg/ml
Environment
Embryo #1
Embryo #2
Embryo #3
Embryo #4
Embryo #5
Figure 1: Graph showing the control (recorded in vivo and in vitro heart
rates of embryos before the application of dobutamine) and experimental
heart rates (bpm) following the exposure to 1.25 x 10-4 mg/ml dobutamine.
Results
180
Average Heart Rate
160
140
120
100
80
153.8
119.2
163
149.8
154.2
60
40
20
0
In Vivo
In Vitro
0.000125mg/ml
0.00125mg/ml
0.0125mg/ml
Environment
Figure 2: The average heart rate of five 8-day chicken embryos following
exposure to dobutamine at the specified concentrations. In vivo and in
vitro controls are also displayed.
200
182
150
150
135
150
95
100
0.0125mg/ml
0.00125mg/ml
0.000125mg/ml
0
In Vitro
50
In Vivo
Heart Rate (bpm)
Results
Environment
Figure 3: Graph displaying heart rates of embryo #1 following exposure to
dobutamine at the specified concentrations. In vivo and in vitro controls
are also displayed. Noted a.) Strong contractions immediately after the
administration of dobutamine b.) Asynchronous with bouts of tachycardia
232
250
200
162
150
235
232
156
100
0.0125mg/ml
0.00125mg/ml
0.000125mg/ml
0
In Vitro
50
In Vivo
Heart Rate (bpm)
Results
Environment
Figure 4: Graph displaying heart rates of embryo #2 following exposure
to dobutamine at the specified concentrations. In vivo and in vitro
controls are also displayed. Noted a.) Large, mature embryo; good
preparation b.) Very strong ventricular contractions c.) Explanted heart:
strong contractions leading to fibrillation.
200
167
150
111
140
110
100
92
0.0125mg/ml
0.00125mg/ml
0.000125mg/ml
0
In Vitro
50
In Vivo
Heart Rate (bpm)
Results
Environment
Figure 5: Graph displaying heart rates of embryo #3 following exposure to
dobutamine at the specified concentrations. In vivo and in vitro controls
are also displayed. Noted a.) Bouts of tachycardia b.) Highest
concentration of dobutamine caused eventual atrial flutter which in turn
led to fibrillation
200
153
150
139
114
134
146
100
0.0125mg/ml
0.00125mg/ml
0.000125mg/ml
0
In Vitro
50
In Vivo
Heart Rate (bpm)
Results
Environment
Figure 6: Graph displaying heart rates of embryo #4 following exposure
to dobutamine at the specified concentrations. In vivo and in vitro
controls are also displayed. Noted a.) Explanted heart utilized for all in
vitro data b.) Weak, normal sinus rhythm c.) At highest concentration,
heart went into atrial flutter
200
172
150
137
158
118
80
100
0.0125mg/ml
0.00125mg/ml
0.000125mg/ml
0
In Vitro
50
In Vivo
Heart Rate (bpm)
Results
Environment
Figure 7: Graph displaying heart rates of embryo #5 following exposure to
dobutamine at the specified concentrations. In vivo and in vitro controls are
also displayed. Noted a.) Explanted heart utilized for all in vitro data b.) Heart
alternated between normal sinus rhythm and atrial flutter after administration
of 1.25 x 10-4 mg/ml dobutamine c.) 1.25 x 10-3 mg/ml: Normal sinus rhythm
d.) 1.25 x 10-2 mg/ml: Normal sinus rhythm with strong ventricular contractions
Results
Only five out of the nine embryos tested gave reliable data.
– One embryo was deformed.
– One had a tumor on its heart.
– Two of the embryos gave poor and inaccurate test results.
Conclusions
• In vivo, normal sinus rhythm was routinely observed for all
embryos. This may be attributed to the fact that the embryos
remained in their natural environment.
• In vitro, the average heart rate decreased due to the embryos’
removal from their natural environment and possible damage
to the extraembryonic membranes.
• Overall, the HR (bpm) increased with the initial application
of dobutamine, 1.25 x 10-4 mg/ml.
• A trend of increased HR (bpm) was observed since the HR
also elevated in the next highest concentration, 1.25 x 10-3
mg/ml dobutamine.
• At the highest concentration, 1.25 x 10-2 mg/ml dobutamine,
the heart rate was inconsistant and arryhthmias such as atrial
flutter, bouts of tachycardia, and fibrillation were routinely
observed.
Future Experiments
http://www.ion.ucl.ac.uk/images/Fluorescence-Microscope.jpg
• Data will be recorded in the form of a timeline using 15second intervals in order to better track irregular heart
beats.
• Only explanted hearts will be used.
• Immunofluorescence of B1-adrenergic receptors
themselves will be performed.
References
•
•
•
•
•
•
•
Cruz, Y.P. (1993). Developmental biology: A guide for experimental study.
Sinauer Associates, Sunderland, Massachusetts.
Driscoll, D.J., Gillette, PC., Lewis, R.M., Hartley, C.J., Schwartz, A. (1979).
Comparative Hemodynamic Effects of Isoproterenol, Dopamine, and
Dobutamine in the Newborn Dog. Pediatric Research, 13, 1006-1009.
Fiser, D.H., Fewell, J.E., Hill, D.E., Brown, A.L. (1988). Cardiovascular and renal
effects of dopamine and dobutamine in healthy, conscious piglets. Critical Care
Medicine, 16 (4), 340-345.
Germann, W.J. and Stanfield, C.L. (2005). “Principles of Human Physiology”
Second Ed. (S. Beauparlant, Editor). Pearson Education, Inc., San Francisco.
153, 443.
McLaughlin, J.S. and McCain, E.R. (1998). “Developmental and Physiological
Aspects of the Chicken Embryonic Heart.” Tested Studies for Laboratory
Teaching, Volume 20 (C.A. Goldman, Editor). Proceedings of the 20th
Workshop/Conference of the Association for Biology Laboratory Education
(ABLE), 20: 85-100.
Sonnenblick, E.H., Frishman, W.H., LeJemtel, T.H. (1979). Dobutamine: a new
synthetic cardioactive sympathetic amine. Medical Intelligence, 300, 17-22.
Tyler, M.S. (1994). Laboratory exercises in developmental biology. Academic
Press, San Diego, California.