Experimental evaluation of anti-arrhythmic drugs

Dr Kirtan Bhatt

Post-graduate,

KIMS, Bangalore

Protocol • Introduction

• Recording of ECG in experimental animals

• Experimental modelsIn vitro modelsIn vivo models

1. Chemically induced arrhythmias2. Electrically induced arrhythmias3. Mechanically induced arrhythmias4. Exercise induced arrhythmias

Genetic modelsCell culture models

• Conclusion

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

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:

• 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

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

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

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

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

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

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

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)

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

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

Ach or potassium induced arrhythmias

• New Zealand white rabbits weighing 0.5-3 kg are used

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 periodFibrillation induced and allowed to continue for

3 minutes

Test compound is added to bath

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.

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

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.

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)

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

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

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

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

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. cephalicaantebrachii)

• Duodenum is cannulated for intraduodenal administration

• ECG is recorded with needle electrodes from lead II. Heart frequency is derived from R-peaks of ECG.

• 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

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

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

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

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

Some other models for chemically induced arrhythmias1. 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)

Electrically induced arrhythmias

1. Ventricular fibrillation electrical threshold

2. Programmed electrical stimulation induced arrhythmia

3. Sudden coronary death model in dogs (Harris dog model)

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

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.

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.

Evaluation:

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

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

• 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.

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.

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

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)

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

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.

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.

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.

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.

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

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.

• 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.

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.

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.

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.

• 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.

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.

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.

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

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.

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.

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