Download Chronotropic Effects of Select Cardiovascular Drugs on the

Chronotropic Effects of Select Cardiovascular Drugs on the Developing
Vertebrate Heart
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Jacqueline McLaughlin, 2Vinod Jeyaretnam
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Penn State Lehigh Valley; 2Undergraduate student
Abstract
An experiment was performed to investigate the effects of two known cardiovascular
drugs on the developing 4-chambered vertebrate heart using the embryonic chicken as the
model system. Ninety percent of pregnant women take some type of drug: whether that
is prescription, over-the-counter, or social in nature. The FDA has developed a grading
rubric to rate a drug that may be used during pregnancy based on the risk that drug poses
to the developing fetus. The human fetus is particularly vulnerable to birth defects
between the third and eighth weeks of development – which directly correlates with the
timing of major heart developmental stages. In the following study, two known
cardiovascular drugs with opposing effects were exposed to isolated chicken hearts at
times during development that denote crucial heart organogenic events in order to
directly study chronotropic effects. Ro 40-5967 is a known potent T-type calcium
channel antagonist, while NKH477 is known to have positive inotropic and chronotropic
effects. The pharmacokinetics and pharmacodynamics of both drugs is fairly well
understood in numerous adult vertebrate model systems; however, no information exists
on the effects of these drugs on a vertebrate embryo. Due to known chronotropic effects
in the adult model system it was hypothesized that Ro 40-5967 would decrease heart rate
while NKH477 would increase it. In this study, the effects of Ro 40-5967 and NKH477
were tested on “isolated” embryonic chicken hearts using the live cell Bioptechs ®
thermal control system, the Delta T-EDU. Baseline heart rates (HR) were determined for
in vivo, in vitro whole embryo, and in vitro isolated hearts. Both drugs were administered
at 10-4, 10-3, 10-2, and 10-1 mg/mL, and at each concentration the average HR was
determined and arrhythmias noted. The proposed hypothesis was not supported. There
was a clear difference between 6-day and 7-day embryos resulting in a “D” and “C” FDA
drug risk grade for Ro 40-5967 and NKH 477, respectively. In conclusion, it is
recommended that neither drug be administered to a pregnant woman.
Introduction
Drugs administered to pregnant mothers have the
potential to cross the placenta and reach the fetus. The
chicken embryo is a classic organism used to investigate
vertebrate heart development and the pre-natal toxicity of
maternally administered drugs. The United States Food
and Drug Administration (FDA) classifies drugs
according to the degree of risk they pose for the fetus.
How a drug affects
Figure 1- EDUSTGE 7522-05-08 of
the Delta T-EDU Bioptechs
apparatus.
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a fetus depends on the fetus’s stage of development and the strength and dose of the drug.
Certain drugs taken early during pregnancy may or may not affect the fetus. However, the
fetus is particularly vulnerable to birth defects between the 3rd and the 8th week after
fertilization, when its major organs are developing. The vertebrate 4-chambered heart,
along with the eyes and brain, is among the first organs to develop in humans and
chickens. A one-month human heart resembles that of a 2.5 – 3- day chick heart; a twomonth human heart resembles that of a 6-day chick embryo. Herein, 6-day and 7-day
chick embryos were tested to simulate a developing human heart system during known,
critical heart development events.
Figure 2. Comparative embryology of the 60-hour chicken
model system (left) and a one-month-old human (right).
6-day and 7-day Results
According to previous research conducted on adult vertebrate model systems such as
hamsters, rats, dogs, and humans, Ro 40-5967 has been shown to decrease HR linearly as
concentration increases. This is clearly evident in the 7-day heart, but not the 6-day heart
suggesting that the embryonic pacemaker, the sinus venosus, is still controlling heart rate
at 6-days and reacts differently than the mature sinoatrial node at 7-days to this drug.
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Ro 40-5967 6-day
Average Heart rate (bpm)
170
150
130
y = -20.833x2 - 121.77x - 32
110
90
70
50
-6
-4
-2
0
Log of Drug Concentration (mg/mL)
Ro 40-5967
CMRL 1066 1X
Poly. (Ro 405967)
Figure 3. A scatter plot depicting the heart rate of 6-day “isolated” chicken hearts.
CMRL 1066 1X was used as the control. An increase in heart rate (bpm) was observed at
the three lowest concentrations of Ro 40-5967. The highest concentration saw a dramatic
drop in bpm. Atrial flutter was also observed.
Average Heart Rate (bpm)
Ro 40-5967 7-Day
y = -8.4x + 58.833
130
120
110
100
90
80
70
60
50
-6
-4
-2
0
Log of Drug Concentration (mg/mL)
Ro 40-5967
CMRL 1066 1X
Linear (Ro 405967)
Figure 4. A scatter plot depicting the bpm of 7-day “isolated” chicken hearts. CMRL
1066 1X was used as the control. A linear decrease in bpm was observed. Atrial flutter
was observed at the highest concentration of Ro 40-5967.
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NKH477 6-Day
160
Average Heart Rate (bpm)
150
140
130
y = -13.792x2 - 84.842x + 21.125
120
110
100
90
NKH477
CMRL 1066 1X
Poly. (NKH477)
80
-6
-4
-2
0
Log of Drug Concentration (mg/mL)
Figure 5. A scatter plot depicting the bpm of 6-day “isolated” chicken hearts. CMRL
1066 1X was used as the control. An increase in bpm was observed over the three lowest
concentrations. The highest concentration saw a slight drop in bpm.
Average Heart Rate (bpm)
NKH477 7-Day
150
145
140
135
y = -2.75x2 - 6.45x + 143.58
130
125
120
115
NKH477
CMRL 1066 1X
Poly. (NKH477)
110
-4.5
-2.5
-0.5
Log of Drug Concentration (mg/mL)
Figure 6. A scatter plot depicting the average heart rate of day seven “isolated” chicken
hearts. CMRL 1066 1X was used as the control. An increase in heart rate was observed.
The highest concentration saw a slight drop in bpm.
Conclusion
Overall, these data suggest that Ro 40-5967 and NKH477 have opposing effects on
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embryonic vertebrate heart rate, and neither should be used in a clinical setting until
proper dosage and deleterious effects are further analyzed. Data also strongly suggest that
the affect of these drugs on the developing heart depends on the embryo’s stage of
development and the strength and dose of the drug.
Based on the data collected and the rubric for grading FDA drug risk during pregnancy,
the following ratings for each drug are suggested:
• Ro 40-5967 gets a “D” because studies in humans, or investigational or post
marketing data, have demonstrated fetal risk. Nevertheless, potential
benefits from the use of the drug may outweigh the potential risk.
• NKH477 gets a “C” because risk can not be ruled out. Adequate, wellcontrolled human studies are lacking, and animal studies have shown a risk
to the fetus or are lacking as well. There is a chance of fetal harm if the drug
is administered during pregnancy; but the potential benefits may outweigh
the potential risks.
Future experimentation will be conducted to further understand when, exactly, the
intrinsic conduction system of the chicken heart is fully developed, and how these and
other cardiovascular drugs directly effect the Ca+2 based, T-type channels that regulate
the “pace-maker” potential.
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