A presentation describing in vitro and in vivo methods for experimental evaluation of anti-arrhythmic drugs

1. Experimental evaluation of
anti-arrhythmic drugs
Dr Kirtan Bhatt
Post-graduate,
KIMS, Bangalore

2. Protocol
• Introduction
• Recording of ECG in experimental animals
• Experimental models
In vitro models
In vivo models
1. Chemically induced arrhythmias
2. Electrically induced arrhythmias
3. Mechanically induced arrhythmias
4. Exercise induced arrhythmias
Genetic models
Cell culture models
• Conclusion

3. Introduction and historical landmarks
1897 – Oskar/Oscar Langeddorrf
1975 – Laszlo Szekeres, Papp
1979 – Laszlo Szekeres
1984 – Winslow
1984 – Wilson
1988 – Walker
1993 – Cheung
Laszlo Szekeres

4. Recording of ECG in experimental animals
• Recording of the ECG is an essential tool in the evaluation of anti-
arrhythmic drugs. The pattern of ECG varies between species.
• Some changes:
Lead II between right foreleg and left hindleg, which is in line with neutrally
placed heart
Lead I between right and left foreleg stated to lie in the axis of horizontal
heart
Lead III between left foreleg and left hindleg in line with the vertical heart.
Along with these, unipolar leads (V1 to V6) and aVL, aVR and aVF.
• The procedure for recording of ECG in rats is as follows:

5. • Male Sprague Dawley rats 250-300 g
anaethetised by IP pentobarbitone 50 mg/kg
• Right jugular vein – injections and left
coronary for BP
• Lead II
• Electrode constructed with 26 gauge needles
placed subcutaneously
• Paper speed 100 mm/s on a student
physiograph
• Sensitivity of student physiograph adjusted
to provide deflection of 30 mm for 1mV
standard square wave

6. ECG recording (cont…)
• Variables measured:
 σ T
 P-R interval
 QRS
 Q-T
 RSh
• Some differences between ECG of humans and smaller animals
 Heart rate is very high
 More prominent QRS
 ST segment is generally absent

7. In vitro methods
• Isolated Guinea Pig papillary muscle
1. Characterization of anti-arrhythmic activity
2. Action potential and refractory period
• Lagendorff technique
• Ach or potassium induced arrhythmias

8. Isolated guinea pig papillary muscle
• A simple and accurate method to classify anti-arrhythmic drugs into
class I, II, III and IV.
• Based on the changes seen in
– Tension developed in papillary muscle (DT)
– Excitability (EX)
– Effective refractory period

9. Guinea pig (200-400 g) stunned and carotid artery severed
Thoracic cage opened and heart removed
Myocardium placed into a container filled with pre-oxygenated and prewarmed PSS
Right ventricle opened and tendinous end of papillary muscle ligated with a silk thread (connected
to a force transducer ).
Chordae tendinae freed from the ventricle and the other end clamped into a tissue holder and
platinum wire electrodes attached at its end
Preparation transferred to PSS and maintained at a constant temp and pressure

10. Muscles are field stimulated to contract
isometrically at stimulus duration of 1 ms at 1 Hz
frequency
Pulses delivered through a Grass constant voltage
stimulator and tension recorded through a
polygraph
Force frequency curve obtained by measuring
developed tension over a range of stimulation
frequencies
Percentage change in developed tension, ERP and
the strength duration curve noted

11. Action potential and refractory period in Guinea
pig muscle
• Guinea pigs of Marioth strain of 250-300 g
• Two strongest papillary muscles from LV taken
• Standard microelectrode technique is used to measure AP
• Stimulation is given by rectangular pulses of 1 V and 1ms duration at
500ms interval
• Second stimuli given in decremental intervals till contraction ceases

12. Lagendorff technique
Principle
• The heart is perfused in a retrograde direction from the aorta either
at a constant pressure or a constant flow with oxygenated saline
solutions
Animals
Albino rats (300g and at least 1 year of age)
New Zealand rabbits (1.5-3 kg and 3 years of age)
Guinea pig (300-450 g and 2-3 years of age)

13. Animal housing conditions
- Housed at ambient temperature (23⁰C ± 2⁰C)
- 12:12 hour light and dark cycle
- Free access to tap water
- Food ad libitum
Precautions
- Pretreatment with heparin
- Maintenance of PSS flow rate to prevent edema of cardiac tissue
- Prevent air bubble entry
- Maintain sufficient hydrostatic pressure by maintaining distance between
heart and the PSS reservoir
- Cannula shouldn’t penetrate the aortic valve

15. Guinea pigs sacrificed by
stunning
Heart removed as
quickly as possible and
placed in a dish
containing PSS at 37⁰C
Cannula inserted into
aorta and tied and the
heart is perfused with
oxygenated PSSsolution
Oxygenated PSS solution
perfused at a constant
pressure of 40 mmHg at
37⁰C
Test compound
administered
Epicardial ECG electrode
used for pulsatile
stimulation and
arrhythmia induction
Steel hook with a string
is attached to the apex
FOC measured
isometrically by a force
transducer and recorded
on a polygraph
Incidence and duration
of VF or VT recorded in
both test and control
groups

16. Ach or potassium induced arrhythmias
• New Zealand white rabbits weighing 0.5-3 kg are used

17. New Zealand white
rabbits sacrificed and
heart removed quickly
Atria dissected and
attached to an
electrode in lower part
of bath and suspended
Fibrillation produced
when atria exposed to
Ach 3*10-4 or KCl 0.10 g
After 5 minutes of
exposure, rectangular
pulses given (0.75 ms
duration, 10V and)
Controlled arrhythmias
are produced and
allowed to continue for
6-10 minutes
30 minutes rest period
Fibrillation induced and
allowed to continue for
3 minutes
Test compound is
added to bath

18. Chemically induced arrhythmias
Chemical agents capable of producing arrhythmias are:
Anaesthetic agents like chloroform, ether, halothane (sensitizing agents)
followed by a precipitating stimulus such as IV adrenaline, aconitine, cardiac
glycosides (ouabain), veratrum alkaloids.
The sensitivity of these arrhythmogenic substances differs from species to
species.

19. Various models for chemically induced arrhythmias are:
1. Aconitine induced arrhythmia in rats
2. Digoxin-induced arrhythmia in Guinea pigs
3. Strophanthin/Ouabain induced arrhythmia in dog
4. Adrenaline induced arrhythmia in dogs
5. Calcium-induced arrhythmia in Wistar albino rats

20. Aconitine induced arrhythmia in rats
• Aconitine is a plant alkaloid - Aconitum
napellus root
• Can persistently activate sodium channels -->
ventricular arrhythmia in anaesthetised rats.
• Drugs with anti-arrhythmic property can be
tested in aconitine-intoxicated rats.

21. Procedure:
Animal – Male Ivanovas rats weighing 300-400 g
Anaesthesia – Intraperitoneal injection 1.25 g/kg urethane
• 5 μg/kg aconitine dissolved in 0.1 N HNO3 is infused into saphenous vein at
0.1 ml/min
• Lead II is recorded every 30 seconds
• Test compound given – oral or IV (3 mg/kg 5 minutes before aconitine)

22. Evaluation
The anti-arrhythmic effects are measured by amount of aconitine/100g
(duration of infusion) which includes
• Ventricular extra-systoles
• VT
• VF
• Death
Statistical significance is assessed by student’s t-test

23. Digoxin induced ventricular arrhythmias
• Overdose of cardiac glycosides can cause ventricular extra-systoles, VF
and death
• The occurrence of these symptoms can be delayed by anti-arrhythmic
drugs
Procedure
Animal – Male guinea pig (Marioth strain) weighing 350-500 g
Anaesthesia – 35 mg/kg pentobarbital sodium intraperitoneal

24. Trachea, a jugular
vein and a carotid
artery catheterised
Positive pressure
ventilation given with
a respiratory pump
BP monitored in the
carotid
Digoxin infused into
jugular vein 85 μg/kg
in 0.266 ml/min until
cardiac arrest
Treated group
receives the test drug
orally/IV 1 minute
prior to infusion
Control group
receives only digoxin
Period till ventricular
extra-systoles, VF and
cardiac arrest is noted

25. Evaluation
Doses of digoxin required to induce ventricular extra-systoles or VF or
cardiac arrest after treatment with anti-arrhythmic drugs are compared
statistically with student’s t-test

26. Strophanthin/Ouabain induced arrhythmia in dogs
Animal – Male or female dogs weighing 20 kg approximately
Anaesthesia – IV pentobarbital sodium 30-40 mg/kg
• Two peripheral veins are cannulated for administration of arrhythmia
inducing substance (V. brachialis) and for test substance (V. cephalica
antebrachii)
• Duodenum is cannulated for intraduodenal administration
• ECG is recorded with needle electrodes from lead II. Heart frequency
is derived from R-peaks of ECG.

27. • Strophanthin K is given continuous IV infusion at 3μg/kg/min.
• Signs of intoxication in the form of VT or multifocal ventricular arrhythmias
are seen in 30-40 minutes.
• Infusion is stopped. When the arrhythmia is stable for 10 minutes, the test
substance is given IV 1-5 mg/kg or intraduodenally 10-30 mg/kg.
• ECG is recorded at -0.5, 1, 2, 5 and 10 minutes following the test drug
administration

28. Evaluation:
For IV test drug administration
- Considered anti-arrhythmic if extra-systoles disappear immediately
- Increase the dose every 15 minutes if they don’t
For ID test drug administration
- Considered anti-arrhythmic if extra-systoles disappear in 15 minutes
- “no effect” if it doesn’t improve intoxication in 60 minutes

29. Calcium induced arrhythmias
Al-Obaid et al in 1998 used calcium chloride induced arrhythmias for
anti-arrhythmic activity evaluation in anaesthetized male rats
Wistar albino rats weighing 60-130 g are used
Anaesthesia – pentobarbital 60 mg/kg intraperitoneal

30. Arrhythmia induced
by a single IV
injection 10% CaCl2
(50 mg/kg)
Recovery allowed for
15-20 minutes
Test compound is
administered at
different doses IV
Effect of test
compound on basal
HR noted
After 7 minutes,
CaCl2 re-
administered
Effect of treatment
on induced
arrhythmia noted as
percentage change

31. Adrenaline induced arrhythmias
• Adrenaline can precipitate arrhythmia at high doses
• Dogs of 10-11 kg are anaesthetized by pentobarbitone sodium 30-40
mg/kg intraperitoneally.
• Adrenaline is given through femoral vein at 2-2.5 mg/kg
• Lead II ECG and atrial ECG are recorded
• Test drug given 3 minutes after adrenaline infusion

32. Some other models for chemically induced
arrhythmias
1. Mouse chloroform model (Lawson, 1968 and Vargafting, 1969)
2. BaCl2 model (Papp et al, 1967)
3. Benzene vapours induced arrhythmia (Tripathi and Thomas, 1986)
4. Wenzel and Kloeppel demonstrated that arrhythmias could be
induced by changing the medium of cultured heart cells
5. VF production by isoprenaline and COMT inhibitor at high
temperature (Sono et al, 1985)
6. Grayanotoxin – I induced arrhythmia in guinea pigs (Takei et al,
1994)

33. Electrically induced arrhythmias
1. Ventricular fibrillation electrical threshold
2. Programmed electrical stimulation induced arrhythmia
3. Sudden coronary death model in dogs (Harris dog model)

34. Ventricular fibrillation electrical threshold
Several electrical stimulation techniques are used to measure VF
threshold:
• Single pulse stimulation
• Train of pulses stimulation
• Continuous 50 Hz stimulation
• Sequential pulse stimulation
Animals – adult dogs weighing 8-12 kg
Anaesthesia – sodium pentobarbital 35 mg/kg intraperitoneal

35. Ventilation given with
Harvard respiratory
pump, systolic BP
monitored and body
temperature maintained
with a thermal blanket
Chest opened by a
midline sternotomy and
heart suspended in a
pericardial cradle
Sinus node crushed and
a 2mm diameter Ag-AgCl
stimulating electrode
embedded in a Teflon
disc sutured to ant wall
of LV
3-ms square anodal
constant current pulses
given for 400ms of basic
cycle and prematurely
restimulated
Recording electrode is
placed on the surface
of each ventricle
silver plate is implanted
under the skin in the
right femoral region as
indifferent electrode.

36. Lead II of the body surface
ECG is monitored.
To determine VF threshold, a
0.2-1.8 second train of 50 Hz
pulses is delivered 100ms
after every 18th basic driving
stimulus.
The current intensity is
increased from the diastolic
threshold in increments of
10μA to 1.0 mA or until VF
occurs.
Defibrillation given when VF
occurs,heart allowed to
recover to control conditions
for 15-20 minutes. Anti-
arrhythmic drugs are given in
the femoral vein.

37. Evaluation:
VF threshold is determined before and after test drug administration
and compared using student’s t-test.

38. Programmed electrical stimulation induced
arrhythmias
A ligature is tied around the
artery and needle. The
needle is then removed
which causes stenosis of the
vessel. LAD is perfused for 5
minutes.
Ischemic injury is achieved by
2 hour occlusion of LAD and
then again vessel is perfused
for 2 hours in presence of
stenosis.
During reperfusion, an
epicardial bipolar electrode is
sutured to the IV septum,.
Silver disc electrodes are
implanted SC for ECG
monitoring.
Animals with sustained VT
and VF are taken for study.
HR, ECG are recorded before
the PES is started.
After 6-9 days, the chest is
reopened and PES is
performed through electrode
implanted on non-infarcted
zone with pacing stimuli set
at 200ms.
After 15 pacing stimulation,
an extra stimulus is given

39. • Test drug is given 30 minutes after the stimulus.
• The minimum intensity of current needed for sustained VF is recorded
before and after test drugs and mean values of 10 experiments are
compared by student’s t-test.

40. Sudden coronary death model in dogs
• The group of Lucchesi described the experimental dog model for
protection against sudden coronary death.
Animal - Male Mongrel dogs of 14-22 kg weight
Anaesthesia - pentobarbital sodium 30 mg/kg IV.

41. Direct anodal 15μA current
from 9-V nickel-cadmium
battery passed through a 250
ohm resistor and applied to
electrode in the lumen of LCX.
Cathode connected to a SC
implanted disc electrode and
lead II ECG recorded for 30
seconds every 15 minutes on
a cardio-cassette recorder.
The animals are sacrificed
after 24 hours of constant
anodal current of at the
occurrence of VF.
Heart removed and thrombus
mass in LCX is removed,
weighed.
Heart sectioned and stained
with tetrazolium triphenyl
choride (TTC).
Time of onset of ventricular
ectopy and lethal arrhythmia
recorded using the cassette.
Non-sustained and sustained
tachyarrhythmia are
evaluated

42. Mechanically induced arrhythmias
Arrhythmias can be induced by directly by ischemia or and also by re-
perfusion. Studies involving both the mechanisms to produce
arrhythmias have been demonstrated.
1. Reperfusion arrhythmias in rats
2. VF after coronary occlusion and reperfusion in dogs
3. Two stage ligation in dogs (The Harris dog model)

43. Reperfusion arrhythmias in rats
• Ligation of left main coronary artery results in ventricular arrhythmias
and MI.
• ECG is recorded during ligation and also during reperfusion.
• The infarcted tissue is measured by tetrazolium triphenyl chloride
staining

44. Sprague Dawley
rats (350-400 g)
are anaesthetised
with
pentobarbitone
sodium 60 mg/kg
IP.
The animal is
maintained on
artificial
respiration, jugular
vein is cannulated
for drug
administration.
BP is recorded
from carotid artery
with the help of a
pressure
transducer
connected to a
polygraph.
Chest is opened
and heart is
exposed. The left
coronary artery is
located and ligated
for 15-90 minutes
(in case of infarct
size studies) and
subsequently
reperfused for 30
minutes.

45. Test drug is given 5 minutes before ligation.
The number of ventricular premature beats, VT and VF
are counted in the occlusion and reperfusion periods.
After the reperfusion period, the animal is sacrificed
and TTC staining is done to measure the infarct size..
Changes in hemodynamic parameters and infarct size in
drug treated animals are compared with control values.

46. Reperfusion arrhythmias in dogs
• Ligation of coronary artery in dogs may lead to increase in HR, LV end
diastolic pressure, BP and ventricular arrhythmias especially in
reperfusion.
• Animal - Dogs of 20-25 kg weight
• Anaesthesia:
thio-butobarbital sodium 30 mg/kg IP
maintained on IV chloralose 20 mg/kg and 250 mg/kg urethane IV followed
by SC morphine 2 mg/kg.

47. Changes in
parameters
(mortality,
hemodynamic and
arrhythmia) in drug
treated animals are
compared with
controls.
Coronary artery is
ligated for 90
minutes. Test
compound is given
20 minutes prior to
ligation and
reperfusion is done
after the ligation
period.
LV end diastolic
pressure and HR are
determined from LV
pressure curves.
Myocardial
contractility is
measured as a rise
of ventricular
pressure.
Femoral artery is
cannulated to
measure BP and
connected to a
pressure transducer
and ECG is recorded
continuously in lead
II.

48. Two stage ligation in dogs (Harris dog model)
• In 1950, Harris found that mortality in dogs after coronary occlusion
with a 2 stage ligation procedure was lower than with 1 stage ligation

49. Dogs of 8-11 kg are anaesthetised by IV injection of methohexitone sodium 10 mg/kg and
maintained on artificial respiration.
Chest is opened and the heart is exposed.
Left coronary artery is ligated in 2 stages. Two ligatures are tied around the artery and the 21
gauge needle.
1st ligature is tied around and the needle, which is removed
2nd is tied tightly around the artery (after 30 minutes ).
After 24-48 hours of ligation, arrhythmias develop and subside in 3-5 days.

50. • Lead II ECG, atrial electrogram and mean BP are measured.
• Test drugs are infused 10 minutes after coronary ligation.
• Changes in mortality, hemodynamics and arrhythmias in treated
animals are compared with controls.

51. Exercise induced ventricular fibrillation
• Billman and his group developed methods to evaluate anti-arrhythmic
drugs for their activity in exercise-plus-ischemia induced arrhythmia
test.

52. Mongrel dogs 15-19 kg are anaesthetised with sodium
pentobarbitone 10 mg/kg IV, chest cavity opened, heart
exposed and supported in a pericardial cradle.
Around the LCX a 20 MHz pulsed Doppler flow
transducer and hydraulic occluder are placed.
A pair of insulated silver wires are sutured to the
epicardial surface of both the left and right ventricular
electrogram.
A pre-calibrated solid state pressure transducer is
inserted into LV and finally a two stage occlusion of LAD
is done.

53. Leads are placed
under the skin to
exit on the back of
the animal’s neck.
3-4 weeks after the
production of
ischemia, the
animal is made to
walk on a motor
driven treadmill
during which
susceptibility to VF
is tested.
3 minute warm up
period - animals
run at 6.4 km/hr
(0% grade).
The grade is
increased every 3
minutes from 0%,
4%, 8%, 12% and
16%.
In the last minute
of exercise, the LCX
is occluded, the
treadmill stopped
and occlusion
maintained for an
additional minute.

54. • Repeated after the test drug and the findings are compared with the
control (saline) group.
• All hemodynamic data (rate of change of LV pressure) are recorded. The
refractory period data, reactive hyperemia response to each occlusion is
averaged and data analysed using ANOVA.
• The effect of intervention on arrhythmia formation are determined by chi-
square test with Yate’s correction.

55. Cell culture techniques
• Ventricular arrhythmias, especially torsades de pointes can be
evaluated using isolated ventricular myocytes.
• Analysis of action potential and patch clamp techniques in isolated
ventricular myocytes helps us to clarify mechanisms underlying
development of torsades de pointes.

56. Guinea pigs (250-350 g) are sacrificed by decapitation and their hearts are
removed and perfused retrodradely through the aorta at a rate of 10 ml/min
with oxygenated calcium free HEPES buffered saline at 37⁰C for 5 minutes.
Perfusion with same solution containing 300 U/ml type II collagenase and 0.5-
1.0 U/ml type XIV protease for 8 minutes
HEPES buffered saline containing 0.2mM calcium chloride for additional 5
minutes.
The cells re-suspended in HEPES buffered saline and stored at 24⁰C.

57. Transmembrane potential is recorded using glass electrodes connected to
the Axoclamp 2A amplifier.
Cells superfused with HEPES buffered saline at a rate of 2 ml/min at 37⁰C.
Passing brief current pulses (1 ms, 1.2 times threshold) through recording
electrode with active bridge current evokes action potentials.
Cells are stimulated at a frequency of 1 Hz during stabilization period and
at a frequencies of 1 and 3 Hz during control and 10 minutes after
superfusion with test drugs at cumulatively increasing drug concentration

58. Four individual AP are
digitally averaged and
measured for each
condition.
For voltage clamp studies,
microelectrodes made
from square bore,
borosilicate capillary
tubing are filled with 0.5
M potassium gluconate, 25
mM KCl, 5 mM K2ATP.
Voltage clamp is
performed using whole
cell recording mode and
cell perfusion is minimized
by maintaining constant
negative pressure on the
electrode using 1 ml
syringe.
Outward K+ currents are
measured during
superfusion of cells at 2
ml/min with calcium free
HEPES buffered saline.
Concentration response
relations are determined
by measuring AP of
currents in each cell
during control conditions
and during superfusion
with 2 successively
increasing concentrations
of a drug.

59. Genetically induced arrhythmias
• A certain breed of dogs – German shepherds – exhibit propensity to
sudden death that occurs due to inherited ventricular arrhythmias.
• Death usually occurs in them during sleep or at rest after exercise or
excitement.
• These dogs can be used for screening of potential anti-arrhythmic drugs.

60. Conclusion
• Species differences do exist in mechanisms producing arrhythmias and no animal can
exactly resemble humans
• However, knowledge acquired from animal studies can help a great deal in devising
therapeutic strategies for both ventricular and supraventricular arrhythmias
• Various animal models can be combined to build novel strategies in management of
arrhythmias