the isolated heart as a model for the study of cardiovascular disease

7/28/2019 The Isolated Heart as a Model for the Study of Cardiovascular Disease

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The isolated heart as a model for the study of cardiovascular disease.

As discussed in an earlier paper1, the isolated perfused small mammalian heart

probably represents the optimal compromise in the conflict between the quantity

and quality of data that can be acquired from an experimental model versus itsclinical relevance - especially in relation to the modelling of ischemia. In

exploiting this preparation to its full potential it is important to consider a

number of key questions each of which will be addressed in the following

sections. In the course of this discussion, a practical guide to perfusionmethodology will emerge.

What are the advantages and disadvantages of the isolated perfused heart?

At a practical level, the isolated heart, especially from small mammals, provides

a highly reproducible preparation which can be studied quickly and in largenumbers at relatively low cost. It allows a broad spectrum of biochemical,

physiological, morphological and pharmacological indices to be measured (see

later). These measurements can be made in the absence of the confounding

effects of other organs, the systemic circulation and a host of peripheral

complications such as circulating neurohormonal factors. This characteristic

may be considered as an investigational advantage in that it allows thedissection of peripheral from cardiac responses or a disadvantage in that it

makes the preparation one step further removed from the in vivo state. Whilst

the fact that the isolated heart is denervated must always be taken into account,this can be turned to advantage allowing the separation of cardiac from

sympathetic and vagal stimulation. Denervation and the absence of other

peripheral factors can often be compensated for; thus, catecholamines or otherneurotransmitters may be included in perfusates and many other peripheral

factors can be added exogenously and in a controlled manner which again can

represent a significant investigational strength. Certainly, the isolated perfused

heart provides an excellent test-bed for undertaking carefully controlled dose-response studies of metabolic or pharmacological interventions.

It must be recognised that, as an ex vivo preparation, the isolated heart is a

constantly deteriorating preparation but nonetheless it is capable of study forseveral hours. The preparation also readily allows the induction of whole heart

or regional ischemia and this can be achieved at various levels of flow.

Similarly, anoxia or hypoxia at various degrees of oxygen deprivation (in the

presence of normal flow) can be easily imposed. The isolated heart preparation

is amenable to reperfusion or reoxygenation at various rates and with various

reperfusate compositions thus providing a powerful tool for assessing many

aspects of ischemia- and reperfusion-induced injury. Arrhythmias are readily

induced and studied, especially in the larger hearts where conduction pathways


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can be mapped and a variety of electrophysiological recordings made. Of

particular importance, the isolated heart preparation allows experiments to be

continued in the face of events (e.g. infarction-induced loss of pump function,

cardiac arrest or arrhythmias) which would normally jeopardise the survival of

an in vivo experiment.

Which species is best for perfusion?

In essence the hearts from any mammalian species (together with non-

mammalian hearts such as those from frogs or birds) may be perfused.

However, although isolated perfusion of large animal hearts such as pigs,

monkeys, sheep, dogs and even man has been reported, these are less frequentlyused. This is probably on account of the high cost, greater variability, large

volumes of perfusion fluids and cumbersome equipment that is required.

Without doubt the most frequently studied heart is that of the rat but there arenumerous reports of studies with other species such as the rabbit, guinea pig,

hamster, gerbil, ferret and mouse.2

The advent of transgenic technology will

undoubtedly result in increasing numbers of studies using murine preparationsand this will require investigators to become adept at using this, as yet, poorly

characterised model. Unfortunately, although the literature contains an

increasing number of studies which employ mouse hearts, the fundamental

characteristics of the preparation (e.g. pressure-volume and calcium-function

relationships) have yet to be completely characterised. Another caution relates

to the high heart rate of the mouse and the miniaturisation of equipment whichrequires investigators to take due account of the frequency-response limitationsof recording equipment.

In practical terms, the rat heart is by far, the best characterised, it is also the

heart most frequently used for more complex perfusion preparations such as

working and blood perfused hearts. In terms of ease of handling, the rat has a

great advantage over smaller hearts such as the mouse where intraventricular

pressure recordings are more difficult. The rat does however suffer from one,

frequently cited, limitation, namely its very short action potential durationwhich can limit its value (in terms of extrapolating to the human) of some

studies of arrhythmogenesis and anti-arrhythmic drugs. Other species, such as

the rabbit, suffer problems with anesthesia and the guinea pig heart differs from

other species in that it is totally collateralised effectively preventing the study of

regional ischemia in this species. Thus, as no ideal species exists, in selecting aspecies for study it is crucial to recognise weaknesses and exploit advantages.

What kind of perfused heart preparation can be used?


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Although a number of other variants exist, isolated perfused heart preparations

are largely based on adaptations of that originally described by Langendorff3 orthe more complex working preparation described by Neely4.

The Langendorff heartpreparation: As shown inFigure 1,

this involves the cannulation of the

aorta which is then attached to a

reservoir containing oxygenatedperfusion fluid. This fluid is then

delivered in a retrograde direction

down the aorta either at a constant

flow rate (delivered by an infusion or

roller pump) or a constant hydrostatic

pressure (usually in the range of 60-100mmHg). In both instances, the

aortic valves are forced shut and the

perfusion fluid is directed into the

coronary ostia thereby perfusing the

entire ventricular mass of the heart,

draining into the right atrium via the

coronary sinus.

Constant flow or constant pressure perfusion?

Depending on the requirements of the experiment, both of the two modes of

perfusate delivery have advantages and disadvantages. In both instances (in the

absence of any imposed ischemia) and depending upon species, the resulting

coronary flow with a blood-free perfusate is often in the range of 8-12

ml/minute/g wet weight of tissue (a value which is several times that of blood

flow in vivo). Whilst constant flow perfusion adds an additional element ofconstancy to an experiment it has the disadvantage that, unlike constant pressure

perfusion, autoregulatory mechanisms are overridden and it does notautomatically alter the amount of perfusate delivered to the whole heart when

there are changes in heart rate or work or when regional ischemia is imposed

(under which circumstance the same volume of perfusate may be forced through

a much smaller perfusion bed). Switching between constant flow and constant

pressure modes of perfusion is not straightforward and, with simple apparatus,may not be feasible within a single experimental protocol. Similarly, blood-

perfused hearts where circulating volumes are small (see later), may not easily

lend themselves to constant pressure perfusion. In order to address this problem,

Shattocket al

5

developed an electrical feedback system designed to control anisolated heart perfused with a peristaltic pump. This system (now commercially
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available through ADInstruments Ltd, Australia) allows the perfuser to switch

instantly between constant pressure and constant flow modes of perfusion,

enabling perfusion pressures and coronary flow to be controlled over a wide

range, and providing a continuous on-line measurement of both perfusion

pressure and coronary flow if desired. This latter feature is of considerableadvantage in studies of vascular function. This system has been usedsuccessfully with hearts from mice, rats, guinea pigs and rabbits.

Using the rat as an example, the general procedure for Langendorff perfusion isas follows:

Excision of the heart from the donor animal: Isolation of the heart requires thedonor to be rendered unconscious prior to excision. Anesthesia can be induced

by inhalation of agents such as ether, halothane or methoxyflurane or injection

(via an intravenous or intraperitoneal route) with agents such as pentobarbitone.Intravenous administration to a conscious animal is usually via a tail vein,

whereas in the anesthetised state a femoral vein would be the preferred route

(the vein being accessed by a small skin incision). An alternative to anesthesiais cervical dislocation or concussion but in both instances there are major effects

on catecholamines and other circulating factors. However, there is no correct or

ideal procedure for rendering an animal unconscious, nor is there an ideal

anesthetic, each has its advantages and disadvantages and these will vary from

species to species. Ether is hazardous as it is highly flammable and an irritant to

the animal and must only be used in a well ventilated area. The most widelyused anesthetic is pentobarbitone, but this is a cardiovascular and respiratory

depressant that can lead to a reduction of cellular high energy phosphates6.

Whatever the choice of procedure (and this may be influenced by local animal

welfare regulations), every effort should be made to minimise stress by keeping

the animal in a quiet environment prior to anesthesia and by minimising

handling. Induction of anesthesia should be as swift as possible, with the

perception of pain completely suppressed (this can be assessed by determining

the animals response to a stimulus such as the pedal withdrawal reflex). Unless

studying lipid or fatty acid metabolism (where heparin has a lipolytic action) itis advisable to administer an appropriate intravenous dose of heparin or anotheranticoagulant, to prevent the formation of thrombi in the excised heart.

Once the animal is anesthetised the heart can be excised. Generally, the

diaphragm is accessed by a transabdominal incision and cut carefully to expose

the thoracic cavity. The thorax is opened by a bilateral incision along the lower

margin of the last to first ribs, the thoracic cage is then reflected over the

animals head, exposing the heart. Some investigators then cradle the heart

between their fingers (it is essential to do this gently to avoid contusion injury)

and then lift the heart slightly before incising the aorta, vena cava and


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pulmonary vessels. Immediately after excision, hearts are usually immersed in

cold perfusion solution (4 degrees C to limit any ischemic injury during the

period between excision and the restoration of vascular perfusion). Some

investigators prefer to cannulate the aorta in situ prior to excision of the heart,

but whichever the preferred procedure, it is important that vascular perfusion bere-established as soon as possible after excision of the heart. With practice andclose proximity of the perfusion apparatus and site of heart excision the entire

process can be accomplished in less than 30 seconds although some

investigators report times as long as 10 minutes (sufficient for unintentionalischemic preconditioning or even stunning!)

7.

Cannulation and re-

establishment of vascular

perfusion: The aortic perfusion

cannula(Figure 2)can beconstructed from a variety of

materials including glass,

plastic or thin walled stainless

steel. The external diameter is

typically similar to, or slightly

larger than, that of the aorta

(about 3mm for a heart from a

250g rat). Several small

circumferential grooves(Figure2A)or asingle flangeis usually

machined into the distal end of

the cannula to prevent the aorta

from slipping off. Some

cannulae (Figure 2B)are heated

with water-jackets to prevent

any unwanted fall in perfusatetemperature as it is delivered to the heart (see later section on the importance of

temperature regulation).

A water-jacketed reservoir, situated above the aortic cannula, contains the

perfusion fluid which is oxygenated via a sintered glass gas distributor (for

bicarbonate-based perfusion fluids 95%O2 + 5%CO2 is normally used). It is

advisable to have the perfusion fluid gently dripping from the aortic cannulaprior to cannulation since this helps minimise the chance of air emboli at the

time the heart is attached to the cannula. Cannulation is aided by cutting along

the aortic arch to open it, thus giving a larger area for cannulation. Hearts

should be held gently between the tips of blunt-ended fine curved forceps,taking care to avoid stretching or ripping of the aortic wall. The aorta is then
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gently eased over the end of the cannula, taking care not to insert the cannula

too far into the aorta since this would occlude the coronary ostia or damage the

aortic valve. Theaorta is then clamped to the cannula with a small blunt artery

clip, whilst a ligature is rapidly tied around the aorta, locking into the grooves;

theartery clip can then be removed. In the case of the flanged cannula the aortais slid down the cannula so that the tie is against the flange. Full flow of

perfusate should be initiated as soon as theheart is mounted on the cannula.

Once the heart is securely attached to the cannula any surplus tissue (such asbits of thymus, fat or lungs) can be trimmed away. Drainage of coronary

perfusate from the right side of the heart via the pulmonary artery should be

unimpeded, however, in the course of cannulation it is possible to accidentally

ligate the pulmonary artery. Thus, to facilitate adequate drainage it is advisable

to make a small incision in the base of the pulmonary artery using small pointed

scissors. Some investigators elect to cannulate the pulmonary artery,particularly if they are interested in measuring A-V differences in pO2 or are

making a separate collection of cardiac lymph. Some investigators also insert a

small drainage catheter in the apex of the left ventricle to prevent the

accumulation of any Thebesian flow in the left ventricular lumen. After its

passage through the coronary vasculature the coronary flow can either be

discarded or collected for analysis; if recirculating perfusion is required the

coronary effluent can be returned (preferably via a 5m filter) to the perfusionfluid reservoir for reoxygenation.

Once cannulation is completed and coronary perfusion initiated, contractile

function and regular heart rhythm will return within a few seconds but it may be

10 minutes or more before maximum function is established. During this time,

various instrumentations of the heart can be undertaken. Most studies require

contractile function to be measured (not only for the provision of baseline data

but also to monitor the stability of the heart and the extent of any disturbances

of cardiac rhythm). Although contractile activity can be assessed via a strain

gauge attached to the apex of the heart or an open tip pressure transducer

inserted into the left ventricle, the preferred procedure involves the insertion ofa compliant intraventricular balloon. These balloons are often made from thinsilicone rubber or domestic food wrap. Not only is this ideal for the

measurement of isovolumic left ventricular function but it is also a convenient

means of measuring heart rate. For the balloon insertion, the left atrial

appendage is removed to provide a clear field of view and a deflated balloon,

attached to a short rigid catheter and pressure transducer, is introduced into the

left ventricle via the mitral valve. The neck of the balloon is then either sutured

into position or firmly attached to the cannula with a small piece of plasticine.

Great care must be taken while inserting and securing the balloon as it is veryeasy to damage the heart - especially small hearts such as those from mice or
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neonatal rats. The balloon is inflated with water from a microsyringe until a left

ventricular end diastolic pressure of between 4-8 mm Hg is obtained (an

excessively high balloon volume should be avoided since it may cause tissue

compression and subendocardial ischemia which in turn will lead to an unstable

preparation with an increased propensity to arrhythmias). Once the balloon is inposition, left ventricular systolic, diastolic, and developed pressure can berecorded. If the heart is to be paced, one silver wire recording electrode can be

hooked on the ventricle with the reference electrode attached to the stainless

steel cannula. A bipolar stimulator is recommended in order to avoid toxicity

from electrolysis of the perfusion fluid which can occur when unipolar

stimulation is used. If an ECG is to be recorded, the electrodes can bepositioned as required, with again, the stainless steel cannula making a suitable

indifferent electrode. Once instrumentation of the heart has been completed, a

temperature regulated heart chamber should be placed around the heart and thetop covered over with domestic food wrap or other thin material.


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The working heart preparation: As shown inFigure 3, this is a more complex

preparation with ventricular filling via the left atrium and ejection in the normal

direction via the aorta. This preparation offers the advantage of an ability to

measure pump function with different filling pressures and afterloads. Rat

hearts are the most frequently used species for working heart preparations but

all species can be used - even the dog or pig.
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Excision of the heart from the donor animal: Since the first step in establishing

a working heart preparation is to set up a Langendorff preparation, theprocedure for anesthesia and excision is identical to that described earlier.

Cannulation and re-establishment of vascular perfusion: Again, the first stepsare identical to the Langendorff procedure described earlier, except that the

Langendorff perfusion line is usually attached to a side arm of the aortic

cannula(Figure 2C).Following the establishment of perfusion(withoutthe

insertion of an intraventricular balloon) there is just one additional step (whichproves most difficult to those learning the procedure) namely the cannulation

and secure tying off, without any leaks, of the left atrium via one of the orifices

of the pulmonary veins. The dimensions and relative positions of the aortic and

atrial cannulae are critical to a successful preparation. Once aortic and left atrial

cannulation are accomplished, the Langendorff perfusion line is clamped ("a"

closed) and perfusion initiated by unclamping the perfusion line from the leftatrium ("c" opened) whilst simultaneously unclamping the aortic outflow line

("b" opened). In this way, oxygenated perfusion fluid from a constant pressure

head left atrial perfusion reservoir (which is continuously filled by a roller pump

from a gassing reservoir which is frequently referred to as the lung) flows

under gravity into the left atrial cannula. The preload of the preparation is

determined by the height of the overflow from the atrial perfusion reservoir

above the heart. This is usually set to 20cm for isolated rat hearts but can be

varied to suit other preparations or to allow construction of "Starling curves"

relating preload to cardiac function. It is important to stress that, in vivo, cardiacoutput is equal to the venous return from the lungs to the left atrium - in the

isolated working heart the venous return is represented by the flow from the left

atrial cannula. An important point, often overlooked by investigators when

constructing a working heart apparatus, is that the left atrial perfusion line must

be capable of delivering perfusion fluid at a rate sufficient to support the

maximum cardiac output of a working heart at any particular preload. If the left

atrial cannula is too small it will artificially limit the cardiac output of thepreparation. The problem is compounded by the pulsatile nature of atrial filling,

which means that the atria only fill during about half of the cardiac cycle. Toensure that this problem does not arise and that left ventricular filling is not

limited by inadequate left atrial inflow it is essential to check that the left atrial

perfusion line can deliver a flow rate of at least twice the expected maximal

cardiac output. This is easily checked by running the apparatus without a heart

attached and measuring the flow from the left atrial line - a rate of at least150ml/min is recommended for a 1g heart. Having flowed from the left atrial

cannula into the left atrium, the perfusion fluid is ejected via the mitral valve

into the left ventricle from where it is ejected through the aortic cannula against

a hydrostatic pressure via the elasticity chamber and flowmeter to the top of thelung. The afterload is determined by the height of the column of fluid above the


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aortic cannula. The elasticity chamber, which contains a known volume of air

for the working rat heart, mimics normal vascular elasticity. It is an essential

component of the perfusion circuit and without it the heart will rapidly fail. In

the course of left ventricular ejection a portion of the perfusion fluid is forced

into the coronary ostia and thereby perfuses the coronary vessels of the heart.The coronary effluent exits from the right heart into the heart chamber fromwhence it may be sampled for assay or returned (via a roller pump) to the lung

for reoxygenation. Depending on the species and experimental design, filling

pressures are usually in the range of 10-20 cmH2O and afterloads in the range

60-100 cmH2O. Under these conditions and using the heart from a 250g rat,

coronary flows of up to 25 ml/minute and aortic flows of 50-80 ml/minute canbe expected. These can be measured by timed collection into measuring

cylinders or by in-line float or electromagnetic flow meters. Summation of

coronary and aortic flow gives the cardiac output. Hearts may be paced or

allowed to beat spontaneously under which circumstances heart rate may be

derived from a pressure recording which is usually via a side arm of the aortic

cannula. Because of the large volumes of perfusion fluid pumped by the heart,

the working preparation usually operates in the recirculating mode and for this

reason it is essential to have an in-line filter (5m porosity) in the circuit to

remove any particulate contaminants which may originate from the heart,connecting tubing, glassware or perfusion solutions.

What is the best perfusion temperature?

Irrespective of whether a Langendorff or working heart preparation is used it is

obviously preferable to perfuse at or near the normal body temperature of the

species under study. This value varies somewhat between species but in general

(unless as in some surgical studies hypothermia is deliberately employed), most

investigators elect to perfuse their hearts at 37.0-37. 5C. It cannot be stressed

too strongly how critical it is (and how difficult it is) to maintain good and

uniform temperature control, it is strongly recommended to have permanent

temperature sensing microthermisters at various parts of the circuit. There are

two basic approaches to maintaining the heart, the perfusion circuit and the fluidit contains at the correct temperature. The first is a thermostatically-regulatedcabinet in which moist warm air is circulated. These are rarely used, they

seldom work well (in part due to the loss of heat that occurs every time the door

is opened) and they can restrict access to the heart. Most investigators choose to

use a thermostatically-controlled water-jacketed system in which all glass

reservoirs, the heart perfusion chamber and as many of the delivery lines as

possible are surrounded by rapidly flowing water at 37.0-37. 5C. To be

effective, this requires a high output, well regulated, water circulator and the

careful design of the circuit since some temperature drop across the apparatus isinevitable. For this reason, key compartments such as the heart chamber, the


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cannula assembly (if water-jacketed) and the Langendorff or left atrial inflow

lines should receive flow from the circulator first. Bifurcation of the water lines

should be avoided as kinking of tubes may lead to different flow rates to

different parts of the apparatus. Finally, investigators should avoid falling into

the trap of setting the output of the circulator athyperthermic temperatures (e.g.39C) in an attempt to compensate for any temperature drop between thecirculator and the heart chamber as this will inevitably cause problems at some

time or another. In general it is far better to suffer a small degree ofhypothermia

in the circuit since this will not damage the tissue and the relationship between

cardiac function and temperature, which, although very steep below 35C, is

relatively flat between 35 and 37C. A key point is to avoid over-heating thetissue and to maintain a constanttemperature even if it is a little below that

desired. Not surprisingly, due to the necessarily very long perfusate delivery

lines, temperature control in hearts that are perfused in NMR spectrometers are

very vulnerable to poor temperature control. Heat loss from the heart chamber

can be reduced by the use of cannulae attached to silicon (notred rubber) bungs

the diameter of which matches that of the heart chamber, thus creating a tight

seal. While this is easy to achieve in a working heart preparation, it is harder in

the Langendorff heart due to the need for balloon insertion, although this can be

overcome by the creation of a small groove in the bung for the balloon catheter.

As mentioned earlier, an alternative approach is to cover the area between asmaller bung and the heart chamber with waxed laboratory film.

What indices of tissue function, integrity and injury can be measured?

As already discussed, the isolated heart, whether it be a Langendorff or working

preparation, provides the opportunity for the acquisition of a very wide

spectrum of highly reproducible data in a rapid and cost-effective manner. A

variety of physiographs can be used to record the data, computer based

recording devices offer some advantages over the more traditional recorders inthat they allow better data storage and analysis.

Morphology and vascular anatomy: The perfused heart can readily be taken forexamination by either light or electron microscopy. In both instances it offers

the very important advantage that fixation can be by coronary perfusion. For

sequential studies, multiple microbiopsies can be taken (starting at the apex of

the heart and working towards the base) at different times during a perfusion

protocol (this necessitates immersion fixation). In addition, whole hearts can be

used for gross morphology such as is required during infarct size studies. Hearts

can also be perfused with a variety of gels, particles and resins that allow thespecific visualisation of vascular perfusion beds.


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Biochemistry: There are a multitude of measurements that can be made using

the perfused heart. Arterio-venous differences in substrates, metabolites such as

lactate, oxygen and a host of other markers of normal and abnormal metabolism

can be made. Leakage of cellular constituents such as enzymes and proteins can

readily be made for the assessment of tissue injury and whole hearts orsequential biopsies can be taken for metabolic analysis - typically of highenergy phosphates and the way in which they are influenced by conditions such

as ischemia and reperfusion. The ability to perfuse hearts in an NMR

spectrometer allows continuous on-line measurement of metabolites and

intracellular ions such as calcium, protons or sodium. Transluminal or surface

reflectance fluorescence spectroscopy allows the continuous monitoring ofintermediates such as NADH or the contractile transients of ions such as

calcium. Suction microelectrodes allow continuous measurement of interstitial

potassium, calcium, pH or monophasic action potentials. The perfused heart

also provides an ideal model for the delivery of vectors in gene transfer studies

where adenovirus or other vectors can be selectively delivered and trapped inthe coronary vasculature.

Cardiac rhythm and electrophysiology: Electrocardiographic recordings allow

the detection, identification and quantification of abnormalities of cardiac

rhythm and microelectrodes allow further analysis - indeed, in larger hearts such

as the rabbit and the pig, conduction pathway mapping and selective ablation ispossible.

Cardiac contractile function: Whereas the Langendorff preparation provides

valuable information on left ventricular systolic and diastolic pressures and their

derivatives, the working heart gives valuable data on cardiac pump function. In

addition, as mentioned earlier, various tension recording devices may also be

attached to the heart. Ultrasonic crystals can be readily used for regional or

transmural function studies and various echo techniques can also be employed

for measurements of wall thickening. Pressure-volume relationships can be

studied with ease as can more rarefied indices of contractile function such as

Emax.8

Pharmacology: Both isolated heart preparations are extremely valuable for

assessing the direct cardiovascular effects of various therapeutic agents in terms

of contractile function, electrical activity or metabolic function. The option for

recirculating and non-recirculating preparations allows drug dose-response

studies to be carried out with great speed and reproducibility and with precise

control over concentration. Another advantage is to be able to rapidly washoutdrugs from the circulation by replacing perfusion fluids.


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Vascular biology: Whilst most research in the isolated perfused heart has tended

to focus on the function and malfunction of the myocyte, using contractile and

metabolic endpoints, it should be stressed that both the Langendorff and

working heart can be used to study vascular reactivity, endothelial and smooth

muscle function and the effect of a variety of interventions on coronary flowand its distribution. Indeed, the isolated heart has been the cornerstone of muchof the work on the no reflow phenomenon.9

Should hearts be paced?

Like so many other facets of heart perfusion, this decision is based on a

compromise between protocol requirements and quality of data. Left to contractat its spontaneous rate, the isolated heart undergoes a small progressive time-

dependent decline in heart rate. Furthermore, spontaneous heart rate in a

perfused heart preparation is usually significantly below the physiological norm.For rat hearts (which have an in vivo rate of 350-400 beats/minute), heart rates

of 250-320 beats/minute, can be expected. This changing baseline will add

additional variability to the data not only as a consequence of the fall in heartrate plus individual-to-individual variability in heart rate but also in the value

for left ventricular pressure which ( depending upon whether the species under

study has a positive or negative staircase) will either increase or decrease as a

consequence of the fall in rate. Some investigators attempt to compensate forthis by expressing function in terms of pressure x rate product.

In vivo, the atria and sino-atrial node are not perfused by coronary vessels but

by extracardiac vessels, which are severed when the heart is excised for

perfusion. The affected tissue is therefore dependent for its oxygen, nutrientsand temperature control upon either: (i) fortuitous superfusion by coronary

effluent flowing out of the right atrium or (ii) deliberate superfusion from a

cannula delivering warm perfusion fluid. The implementation of an atrial

superfusion line certainly helps in maintaining temperature and a more stable

heart rate. However, it complicates the assessment of coronary flow. An

alternative approach is to immerse the entire heart in oxygenated, temperature-regulated perfusion fluid.

Isolated hearts are easily paced to most required levels and in studies where low

heart rates are required (and escape to a higher spontaneous rate may be

threatened), the sino-atrial node may be crushed to prevent natural impulse

generation. Pacing is not recommended in any studies of arrhythmogenesis

(where the pacing may modify the nature or incidence of any arrhythmia) or the

study of agents that influence heart rate. During studies involving severe

ischemia many investigators choose to terminate pacing during the ischemic


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interval and may delay restoration of pacing until several minutes after the onsetof reperfusion.

What is the maximum duration of perfusion?

Perhaps the most frequent question posed by lay visitors to a heart perfusion

laboratory relates to the longevity of the preparation. Clearly, from the momentan ex vivo preparation is established, it will begin to deteriorate and the rate will

depend on a large number of factors including the skill of the operator (avoiding

contusion injury), the species, the composition of the perfusion fluid, the

presence or absence of various drugs, age, heart rate and work load and the

temperature at which the studies are carried out. Certainly, investigators shouldalways undertake their own stability studies and establish their own exclusion

criteria. However, it is our experience that, with both the Langendorff and

working heart preparation, a deterioration of contractile function (e.g. leftventricular developed pressure or cardiac output) of 5-10%/hour can be

expected. However, this can be influenced greatly such that, in some species,

periods of hypothermic arrest with tissue preservation for up to 24 hours orlonger may be followed by a return to approaching initial levels of function. The

rate at which a preparation deteriorates can be critical in the design and

interpretation of some studies where it may be necessary to use time-matchedcontrols with corrections for baseline deterioration when comparing groups.

Should exclusion criteria be used in perfused heart studies?

No self-respecting investigator who uses in vivo preparations would design and

undertake a protocol without pre-defining criteria for the exclusion of

preparations that are, for one reason or another, unacceptable for use.

Unfortunately, only a minority of publications involving heart perfusion report

prospective exclusion criteria or policy for replacement of excluded hearts (thelatter being a potentially difficult issue with the study of arrhythmias10). Those

few publications that do cite exclusion criteria often only state the lower limits

of a functional index (usually spontaneous heart rate or left ventricular

developed pressure), apparently accepting very high values however extreme.

The failure to apply rigorous prospective exclusion criteria in heart perfusionstudies probably has its origins in the remarkable reproducibility of these

preparations - however, this is not an acceptable excuse for ignoring afundamental requirement of good protocol design.

What is the best perfusion fluid composition?

The majority of studies in the literature (with the exception of those involving

NMR where phosphate often has to be removed from the solution) are based ona bicarbonate perfusion fluid as defined by Krebs and Henseleit.11 This


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perfusion fluid, which was supposed to mimic the key ionic content of blood or

plasma and have a pH of 7.4 at 37C, has the following composition (in mM):

NaCl 118.5, NaHCO3 25.0, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, glucose 11.0 and

CaCl22.5. Unfortunately, in formulating this physiological solution, Krebs

and Henseleit failed to take account of the fact that much of the calcium inblood is bound to proteins and the realistic plasma ionisedcalciumconcentration is approximately half of the recommended value of 2.5mM. As a

consequence, for decades, heart perfusers have used excessively high

concentrations of calcium in their perfusion fluid which usually do not contain

protein or molecules capable of binding calcium. This has meant that, in many

studies, hearts have been in a state of continuous inotropic challenge, probablyworking near the upper limits of the calcium/function curve. Nowadays, many

investigators are rectifying this problem by using ionised calciumconcentrations which are in the range of 1.2-1.8mM.

In discussing calcium, it is important to stress that, in preparing perfusion

solutions containing both calcium and phosphate ions, there is a risk of

precipitation of calcium phosphate particles which will occlude coronary

arteries and destroy a preparation. This is readily circumvented in bicarbonate-

buffered solutions by ensuring that the calcium component is the last to be

added and that the pH is lowered by gassing with 95%O2 + 5%CO2 before

adding the calcium. Perfusion fluids should always be filtered (5m filter)

before use to remove the remarkable number of particulate impurities that can

be present in even the purest commercial chemical.

In discussing perfusion fluid composition, it is important to consider the

substrates that are provided to support the large energy requirements of cardiac

contractile function. Traditionally, high concentrations (approximately 10mM)

of glucose are usually added as the sole substrate. The use of diabetic levels of

glucose reflects the fact that normal physiological concentrations of glucose are

unable to sustain adequate function in the perfused heart in the absence of

insulin. Insulin can be added to overcome this problem, however, it is rarely

done - probably because of the other complex cellular effects of insulin. Thechoice of glucose as the sole substrate in most heart perfusion relies on the

hearts ability to utilise almost any substrate as an efficient energy source - this

is despite the fact that, in vivo, fatty acids are the predominant energy source.

The general practice of avoiding fatty acids as an energy source results

primarily from the difficulty of dissolving these agents in aqueous solutions and

the complication of frothing when the fatty acid-containing solutions are gassed.

The same reason, together with cost, probably explains the general reluctance to

add albumin, or other oncotic agents, to perfusion solutions - a decision that

undoubtedly contributes to the severe edema which characterises most perfusedheart preparations. Drugs or other agents can be added to any perfusion


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solutions; this is normally achieved by adding them, in the desired

concentration, to the basic perfusion medium. However, in some cases, (e.g.

unstable compounds) concentrates can be infused via a side arm to the aortic or

left atrial cannula. In this instance, the concentration of the infusate is set so as

to achieve the desired circulating concentration when the infusate is mixed withthe perfusion fluid as it enters the cannula.

What is the most common cause of unstable or failing preparations?

Other than mishandling the heart during excision, making an error in the

formulation of a perfusion solution, adding a toxic agent to a perfusion solution

or the occurrence of arrhythmias, the most common cause of contractile failurein both working and Langendorff hearts is contamination - either particulate or

bacterial. The source of bacterial contamination may be the perfusion apparatus

itself where inadequate washing at the end of an experiment or poor apparatusdesign (small pockets between oversize tubing and a glass or plastic connector

which create spaces where the substrate-containing perfusion fluid can be

trapped) allows bacteria to thrive. Alternatively, the perfusion solution may bethe cause with particulate impurities in reagents or bacterial contamination that

occurs during preparation or storage (it is recommended not to store substrate-

containing solutions for more than a few hours). In both instances the problem

can be alleviated by: (i) filtration (5m filter) in the course of preparation and

inclusion of filters in recirculating perfusion circuits, (ii) making up fresh and

not storing glucose- or substrate-containing buffers which are good bacterialgrowth media and (iii) thoroughly washing the perfusion apparatus with either

detergent or distilled water followed by (with appropriate safety precautions)

boiling water after every day of use. Some investigators resort to the inclusion

of broad spectrum antibiotics in their perfusion media but this is not

recommended. If contamination of the perfusion apparatus does occur it may be

possible to remove it by washing with acid or detergent but usually it is better to

dismantle, wash and sterilise all components. However, a well designed and

properly washed apparatus with well prepared perfusion solutions can be used

for years without contamination occurring.

Oxygen delivery: gassed perfusion solution, perfluorochemicals,

erythrocytes or blood?

Central to the survival of any perfused organ is the continuous provision of

oxygen in quantities sufficient to support normal metabolism, the maintenance

of transmembrane ion gradients and, in the case of the heart, the large amounts

of energy needed to support contraction. In conventional Langendorff and

working heart preparations, oxygen is provided by gassing the perfusion fluid

with high concentrations of oxygen, typically 95%O2 + 5%CO2 (the CO2 being


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required to achieve the correct pH in bicarbonate-based buffers). If there are no

fatty acid or protein additions to the buffer then this is a straight forward

procedure with no complications from foaming. If foaming is a problem then

anti-foam agents can be added (but this is not recommended) or membrane

oxygenators may be employed.

Gassed asanguinous perfusion fluids have a relatively low oxygen carrying

capacity but a very high pO2 (> 500mmHg). The fact that they are able to

provide sufficient oxygen to the heart is testified by: (i) reducing the pO2 byusing gassing mixtures containing only 70% oxygen does not result in a loss of

contractile performance, (ii) the venous pO2 is still relatively high indicating a

reserve in oxygen availability, (iii) perfusion fluid perfused preparations will

work for several hours without any great deterioration in function and (iv) when

challenged with inotropic agents perfusion fluid perfused hearts can increase

their work for sustained periods without injury. However, it should be stressedthat asanguinous perfusion fluids can only provide sufficient oxygen if thecoronary flow rates are several times the physiological norm.

Driven by concerns over the transmission of infections during blood

transfusion, there has been considerable research into developing blood

substitutes and especially perfluorochemical oxygen-carrying hemoglobin

substitutes.These agents, when used as emulsions with aqueous media, are able

to deliver more than twice as much oxygen.12

As a consequence

perfluorochemicals have been used by some investigators in perfusion solutionsbut this practice has not been widely adopted. This probably reflects the general

satisfaction that oxygenated aqueous media are able to deliver sufficient oxygen

to most isolated heart preparations. As a consequence, most work in the

perfluorochemical area has resolved around using these agents to provide

oxygen to tissue which is deprived of adequate flow. In this connection there are

many studies of these agents as additives to cardioplegic solutions or as agents

to limit the evolution of myocardial infarction. However, again, they have notbeen introduced into widespread use.

The artificially high coronary flow rates and high pO2 of asanguinous perfusion

solutions, together with the absence of the many key components of blood,

makes the concept of perfusing isolated hearts with blood an attractive option.

Certainly, blood perfusion is used successfully with many other organs; it would

be expected to allow near normal coronary flow rates; its protein content should

help alleviate the unwanted edema that characterises perfusion fluid-perfused

hearts and it should also allow the heart to be exposed to blood-borne elements

such as neutrophils. The widespread use of blood in heart perfusion has

probably been discouraged by: (i) the volume of blood needed (perfusing one

rat heart would require the blood of several rats), (ii) the inability to use donor


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blood from some (but not all) larger species because they have larger

erythrocytes than the rat and cannot traverse rat heart capillaries and (iii) the

difficulty in oxygenating the blood since conventional gassing creates extensive

foaming and damage to blood cells. However, all of these problems can be

circumvented either through the use of parabiotic preparations with support ratsor the use of membrane oxygenators. As a consequence, a number of blood orerythrocyte preparations have been developed13,14,15,16 and used with greatsuccess.

Blood perfusion: The first report of an attempt to develop an isolated blood

perfused rat heart was published by Gamble et alin 1970.14

A ventilated support

rat was placed in a chamber containing humidified air at 37C and the leftcarotid artery and right jugular vein were cannulated for the supply and return of

blood to an isolated Langendorff heart. Blood from the carotid artery of the

support rat first flowed into a latex bag, the volume of which was maintained

constant by a feedback device. The blood was then pumped to the aortic cannula

of the isolated heart and returned to the jugular vein of the support rat. In 1988,Abraham et al

15modified the preparation by removing the pump.

Blood perfusion with a support animal for oxygenation: The preparation

currently used in our laboratory for rats and rabbits, is based on the procedure

described by Qiu et al13,16, which is a combination of the two methods
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mentioned above. The details for establishing this preparation(Figure 4)in therat are outlined below.

Support rats (350-400g) are anesthetised with sodium pentobarbitone (60mg/kg,

ip) and anticoagulated with heparin (1000 IU/kg). The left femoral artery andright femoral vein are exposed by blunt dissection and cannulated for the

eventual supply and return of blood to an isolated Langendorff perfused heart

from another animal of the same species. An extracorporeal circuit between

these two vessels, primed with a Gelofusine solution (a plasma substituteconsisting of gelatin, sodium chloride and water) is constructed and the flow of

blood through this is gradually established and maintained for a short time

(usually 20 minutes) so as to ensure that the priming solution is adequately

mixed with the blood of the support animal and the entire preparation is stable.

It is advantageous to decrease the hematocrit of the perfusing blood to

approximately 28-30% by mixing it with Gelofusine. This decrease in theviscosity allows for better flow and also helps minimise the possibility of

clotting. The gradual establishment of the extracorporeal circuit is achieved via

an in-line peristaltic pump which is positioned on the arterial outflow of the

support animal and flow through this circuit is gradually increased over 10

minutes to its final required value (which depends on the species used and the

size of the heart but for a 1g rat heart the flow rate would normally be in the

range 2-3 ml/minute). The gradual increase in flow through the extracorporeal

circuit is necessary to prevent the sudden drop in arterial pressure that would

have occurred if the final flow rate of had been established abruptly. Thefeedback system described earlier can be used with this preparation to perfuse

the isolated heart either under conditions of constant flow or constant pressure.

The blood is pumped through a cannula (to which the aorta of the perfused heart

will subsequently be attached) and returned, by gravity, via a reservoir

(comprising of the bottom half of a large syringe positioned in a water-jacketed

heart chamber) and filter (200m filter) to the venous inflow line of the support

animal. Meanwhile, the isolated rat heart is harvested and prepared exactly asdescribed in earlier sections. The heart can also be instrumented with an

intraventricular balloon and other devices exactly as described earlier. Theextracorporeal circuit then flows from the left femoral arterial supply line of the

support animal to the isolated heart. The coronary effluent from the perfused

heart is then returned, by gravity, via a reservoir and filter to the venous inflow

line of the support animal. During periods of global ischemia, the blood can be

diverted from the isolated heart by opening the bypass line (which has beenpositioned to flow into the reservoir) and clamping the line to the heart.

It is possible to artificially ventilate the support rat, however, it is usually

sufficient to allow the animal to spontaneously breathe a mixture of 95% O2 +5% CO2 through a Venturi face mask, the flow rate of which is adjusted to


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maintain blood pO2 and pCO2 within the physiological range. Before placing

the face mask over the support animal, the tongue should be gently pulled

slightly out of the mouth and placed to one side to ensure unobstructed

breathing. Body temperature, monitored by a rectal thermometer, should be

stabilised by means of a thermostatically-controlled heating pad. Blood pressureshould be monitored by a pressure transducer attached to the arterial line. Bloodgas and electrolyte levels of the support rat should also be checked constantly

and corrected when necessary. During the course of the experiment, donor

blood from another rat can be transfused as required to maintain the volume and

stability of the preparation. Additional anticoagulant and anesthetic can be

administered as required.

The above method of blood perfusion requires that very careful attention be

paid to the hemodynamics and blood chemistry of the support animal, with

constant monitoring of vital signs, breathing and the colour of mucousmembranes. An air-filled syringe(Figure 4)above the perfusion cannula acts as

a compliance chamber, which serves to dampen oscillations in perfusion

pressure which occur as a consequence of the contraction of the isolated heartand the peristaltic action of the pump.

Advantages and disadvantages of the preparation: There are a number of major

advantages with this preparation. Firstly, in our experience, it is even more

stable than the isolated perfusion fluid perfused heart, with left ventricular

developed pressure deteriorating less than 5%/hour. The blood perfused heartsuffers far less edema and excellent pressure development is observed (in the

range 130-180 mmHg when end diastolic pressure is in the range 4-8 mmHg).

Coronary flow rate (2-3 ml/minute) is much closer to the physiological range

than that with perfusion fluid perfused preparations and the preparation allows

one to study the effect of blood elements such as neutrophils on cardiac

function. By using two support animals attached to one heart it is possible to

study the effect of transient exposure (or removal) of factors such as

neutrophils. The preparation can be used to study global or regional ischemia at

zero or reduced flow rates exactly as a perfusion fluid-perfused heart and isamenable to the same wide range of biochemical, morphological,

pharmacological and functional assessments. With careful attention to the

support animal, it is our experience that one support rat can be used for several

hours and allow the sequential study of more than one isolated heart.

A potential disadvantages of the support animal method of blood perfusion is

that any substance released by, or administered to, the isolated heart will

normally be transmitted to the support animal with possible deleterious

consequences. On the other hand, in some instances, the support animal may

exert a beneficial action by metabolising or excreting such substances. Another


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related disadvantage of this preparation is that any deterioration of the support

animal will threaten the survival of the isolated heart. However, with care, a

replacement support animal can be introduced into the perfusion circuit. When

perfusing with either whole blood or a washed red cell preparation, care must be

taken to ensure that the perfusate does not come into contact with glass as thiswill promote red cell hemolysis.

Erythrocyte perfusion: If the use of a support animal is not feasible then analternative approach is to undertake isolated heart perfusion with washed red

blood cells using a simple membrane oxygenator constructed from coils of thin

walled silicone rubber tubing. In 1979 Bergmann et al17

reported a preparation

in which isolated rabbit hearts were perfused with perfusion fluid containing red

cells at a hematocrit of 25% or 40%. A key feature of this preparation was theuse of blood cells from a different species (sheep) which are small enough to

traverse the capillaries of the rabbit heart. They found that, in addition to

exhibiting an enhanced stability when compared to crystalloid perfused hearts,

the red cell perfused hearts were less likely to develop significant edema. The

model currently used in our laboratory18

is essentially similar(Figure 5).A red

cell (bovine blood) suspension is kept in a reservoir which is stirred

continuously to prevent red cell sedimentation. From this reservoir the red cellsuspension is pumped in an artificial lung, comprising of gas permeable

silicon tubing wound into a coil. This tubing is housed in a water-jacketed

chamber, which maintains the perfusate at 37C. We find it to be advantageous

to pump the red cell perfusate both into and out of the oxygenator since
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pumping on one side of the circuit can lead to stretching and distortion of the

oxygenator tubing. However, it is essential that the pump speeds are identical to

prevent emptying or pooling of blood in the oxygenator. In addition, 95%O2 +

5%CO2 is pumped into the reservoir so as to maintain oxygenation of the red

cells, whilst keeping pH within the physiological range. The perfusate is thenpassed through a white cell filter, which has two functions: (i) the large surfacearea of the filter and its immersion in heated water provides an efficient method

of temperature regulation and (ii) the filter protects the heart against micro-emboli.

Excision of the heart from the donor animal: The procedure for anesthesia and

excision is identical to that described in the section on blood perfusion with asupport animal.

Preparation of a red cell perfusate: Preparation of a red cell perfusate is moretime-consuming than the preparation of a simple perfusion solution. Fresh blood

(most commonly ovine or bovine) is collected into vessels containing 5,000 IU

heparin/litre of blood and 0.2 MU benzylpenicillin /litre of blood. After filtering(200m filter) the blood is centrifuged at 1000g for 20 minutes, after which the

supernatant and white cell layer are removed and discarded and the red cells

resuspended in nominally calcium-free perfusion fluid. This process is repeated

until the supernatant is clear. If the cells are to be used the next day, it is

advisable to omit the final resuspension, instead storing the packed cells in a

refrigerator (4C) and washing twice on the day of use. Following the finalwash, the red cell concentrate is mixed 1:1 with a dextran/albumin solution,

made by adding 250ml of deionised water to 500 ml of sterile Gentran 70 (a

plasma expander consisting of 6% dextran and 0.9% sodium chloride).

Electrolytes and glucose are added in the following concentrations (mM): NaCl

118.5, KCl 3.8, KH2PO4 1.2, NaHCO3 25.0, MgSO4 1.0 and glucose 10.0. After

filtration (5m porosity) the solution is warmed and 3g of albumin (Fraction V,

Sigma) added. Prior to perfusing, the ionic composition of the solution should

be checked using a blood gas/electrolyte analyser and corrected if necessary

(e.g., the final calcium concentration should approximate the ionised calciumconcentration in blood). Dextran is used to prevent microagglutionation andgentamicin (2 mg/litre) can be added to retard bacterial growth.

Advantages and disadvantages of the preparation: The advantages with this

preparation are similar to those for the blood perfused preparation which uses a

support animal: (i) it is more stable than the isolated perfusion fluid perfused

heart, with pressure development remaining stable for extended periods of

perfusion (approximately 5 % loss/hour), (ii) the red cell perfused heart suffers


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far less edema than the perfusion fluid perfused heart, and (iii) coronary flow

rate (2-3 ml/minute) is much closer to the physiological range than that with the

perfusion fluid perfused preparation. Pressure development in the erythrocyte

perfusion preparation is usually not as great as that seen in the blood perfused

heart and is similar to that seen in the buffer perfused heart.

Perfusing hearts with blood from another species raises the possibility of an

adverse immunological reaction, but as the blood is normally depleted of white

cells and plasma proteins during its preparation, significant reactions areunlikely. However, progressive hemolysis of the red cells during use is

unavoidable, care should be taken to avoid contact with glass (use plastic only)

during the preparation or usage of the blood, as this will greatly accelerate redcell haemolysis.

How can ischemia be best induced?

Whether perfused with an asanguinous solution, washed red cells or blood inthe Langendorff or the working mode, many investigators use the isolated heart

for the study of regional or global ischemia. The isolated preparation is ideally

suited to such studies since pump failure or lethal arrhythmias do not

necessarily terminate an experiment as would be the case with in vivo studies.

Global (whole heart) zero-flow ischemia is readily induced in the Langendorffand the working heart simply by occluding the perfusion inflow lines. Graded

whole heart ischemia at various degrees of flow can also be readily induced in

the Langendorff preparation but is difficult to achieve in the working heart (as a

consequence, investigators often switch the working heart temporarily back to

the Langendorff mode for such treatment). Regional ischemia can also be

induced in both preparations by ligating a coronary artery (usually the left main)and the size of the ischemic zone can be influenced by the positioning of the

occlusion point. To prevent tearing of tissue and also to facilitate reperfusion,

the occluding ligature is usually tied against a small length of plastic tubing.

Reperfusion can be achieved by cutting the ligature. The relatively uniform

coronary artery anatomy of the rat heart offers a great advantage in thatischemic zones of very similar sizes are usually created. The size of the

ischemic zone can be easily estimated by the percent fall in coronary flow (in

constant pressure perfusions) or increase in coronary vascular resistance (in

constant flow preparations). Validation of occlusion is often made by transient

infusion of a coloured dye or fluorescent particles. In the Langendorff heart a

novel dual lumen perfusion cannula19

(Figure 2D)can be used to facilitate

independent perfusion of the left and right coronary ostia. This cannula is a

powerful tool for investigating the regional effects of drugs - it also offers the


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unique opportunity of creating regional low flow ischemia in the rat heart.

Isolated hearts also provide the opportunity for studying hypoxia andreoxygenation, again either regional or global.

Concluding comments.

Having selected the isolated perfused heart as a model to investigate acardiovascular phenomenon, the choice of preparation is very wide. Blood

perfused or perfusion solution perfused, Langendorff or working, each has its

own advantages and disadvantages, both of which can be established in the

quest for a better understanding of cardiac function and malfunction. Although

the investigative power of the preparation is great, as with all experimentalmodels, they are fraught with potential pitfalls, which must be recognised and

addressed. This article has attempted to address many of these issues in the hope

that it will assist investigators to make the best possible use of this experimentalmodel.

the isolated heart as a model for the study of cardiovascular disease

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