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Isolated Perfused
Heart
Radnoti Glass Technology
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Presented by:
Radnoti Glass Technology, Inc.
2007
The Radnoti Working Heart System
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Isolated Perfused
Heart
Radnoti Glass Technology
800-428-1416
The Isolated Perfused Heart System, as originated by Oscar Langendorff more than a century ago, has become a
predominant technique in pharmacological and physiological research. The technique allows the examination of
cardiac contractile strength (inotropic effects), heart rate (chronotropic effects) and vascular effects without the
complications of an intact animal model. From its simple beginning the technique and equipment has evolved to
include both constant pressure and constant flow models in a working heart mode as well as both recirculating
and non recirculating modes. The Radnoti Isolated Perfused Heart System has the capacity to function in any of
these configurations allowing flexibility in experimental research and design.
This presentations intention is a quick reference to instrumentation of the Radnoti Isolated Working Heart System.
Introduction
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Heart
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There are a great number of physiological parameters that can be measured in the Isolated Perfused Heart preparation.
A pressure measurement while in constant flow mode will show the resistance of the heart, indicating vasodilatation or vasoconstriction.
A left ventricular pressure measurement made using a combination of pressure transducer, flexible balloon catheter and latex balloon can serve as an indicator of contractile force. Electrocardiograms (ECGs) are readily obtained using surface electrodes of monopolar, bipolar construction or needle electrodes that pierce the heart muscle and are of interest in studies involving arrhythmias.
Oxygen consumption can be determined with dual oxygen electrodes, one placed in the perfusate stream entering the heart, the other monitoring the effluent leaving the coronary sinus. This effluent can be removed through the use of a peristaltic pump and then transferred to the second oxygen electrode.
Similarly, ion selective electrodes can be placed in the effluent or perfusate stream or oxygenation chamber of the Radnoti Isolated Perfused Heart apparatus thus permitting measurement of pH and other cations and anions.
Radiolabelled compounds can be used for metabolic studies, as well as the release or uptake of various ions or substrates.
Optical studies measuring intracellular constituents have been performed on the fluorescence of endogenous or exogenous fluorescent compounds.
Experimental Options
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Heart
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Perfusion Pressure and Ejection Pressure159905
Perfusion pressure on Aortic Cannula (Langendorff constant flow)
Ejection Pressure on stopcock of Aortic Cannula feed to Compliance loop (Working Heart)
LVP 159907 IVP transducer & Balloon or 159905 Pressure Transducer with 170424 flexible balloon catheter and balloon
Instrumentation of the System
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Heart
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ECG #140155 ECG electrodes and ML132 Bioamp
Flow-
PerfusionIUF1000 Flow Meter mounted at feed to atrial cannulaEjectionIUF1000 Flow Meter mounted inline on compliance loop
Pacing#140157 Pacing electrodesGR-SD9 Stimulator
Instrumentation of the System cont.
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pHMI-410 Micro-combination pH microelectrodeMV-ADPT Millivolt Adapter
O2 MI-730 Dip-type O2 microelectrodeO2-ADPT Oxygen Adapter
TemperatureBAT-12 Microprobe Thermometer
Flexible Balloon Catheter#170423 The balloon catheter is for ventricular insertion. It is a simple, reliable way to measure left ventricular isovolumetric pressure.
Instrumentation of the System cont.
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These values are obtained from a variety of sources and are displayed to demonstrate the approximate ranges of these values. Values are for adult animals. In vivo heart rate and blood pressure are taken at rest. Cation values are from serum. Left ventricular volume (LVV) is given for a balloon inserted into the left ventricle. CF (coronary flow) is given for a saline solution at 50-60 mmHg
Typical Values
Rate Bpm BP mm/Hg Na,mM K,mM Ca,mM Mg,mM LVVml heart Heart
Cat 110-140 125/70 163 4.4 1.3 0.7 0.7-2.4 2-3
Rat 330-360 129/91 140 5.7 2.6 1.1 0.1-0.2 8-10
Guinea Pig 280-300 120/170 145 7.4 2.6 1.2 0.1-0.2 5-8
R.pipens 37-60 31/21
Carp 40-78 43
Rabbit 205-220 110/73 155 4.6 3.5 1.6 0.4-0.7 2-5
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Perfusate or Buffer Delivery: PERISTALTIC PUMP
PERISTALTIC: The Peristaltic Pump is used to transport the buffer solution from the reservoir through the system and to the heart. It is important that the buffer delivery pump offer a range of flow well within the flow demand of the system. The pump should be operating at a mid range of its speed capability to insure a long pump life. The Peristaltic Pump provided with the Radnoti 120101BEZ Isolated Working Heart System is the 170100A Peristaltic Pump with Two 170110 easy load pump heads:
View Instruction Manual
Water Jacket Temperature Control: THERMAL CIRCULATING PUMP
THERMAL CIRCULATOR: The Thermal Circulator is used to warm and maintain temperature of the system by warming the water and circulating throughout the water jacket of the system. The thermal circulator must have sufficient pump strength to move the water through the system and overcome the hydrostatic pressure head created by the elevated components of the system. In addition, the tank volume must be of sufficient size to minimize the effect of the returning fluids’ temperature variation. The combination of these two features will insure an accurate and stable temperature control throughout the system. The thermal circulator provided with the Radnoti 120101BEZ Isolated Working Heart system is the 170051A Thermal Circulating Water Bath
View Instruction Manual
Pumps Overview
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Installation
Locate the unit on a sturdy work area. Ambient temperatures should be inside the range of +50°F to +104°F (+10°C to +40°C).
The maximum operating relative humidity is 80%. Never place the unit in a location where excessive heat, moisture, or
corrosive materials are present. Make sure the voltage of the power source meets the specified voltage, ±10%. The pump
connections are located at the rear of the pump box and are labeled and These connections are angled
upward so the recirculating fluid will drain back into the reservoir when the hoses are disconnected. Both connections are
capped with stainless steel serrated plugs. The pump lines have ¼" MPT for mating with standard plumbing fittings. For your
Convenience, stainless steel adapters ¼" FPT to 3/8" O.D. serrated fitting are provided. The bath work area has a high and
low level marker to guide filling. The markers are 1 inch horizontal slits located in the center of the stainless steel baffle
separating the work area and the pump assembly. The correct fluid level falls between these two markers. The unit will not
start if the fluid level is below the lower slit.
Operation
Before starting the unit, double-check all electrical and plumbing connections. Make sure the bath is properly filled with fluid.
To start the unit, press To turn the unit off press again. The LED indicates the status of the heater. It
illuminates to indicate the heater is on.
Temperature Adjustment
To display the temperature set point simply press on the controller. The indicator will illuminate and the display will flash the
current set point value. To adjust the temperature set point, press the arrow buttons until the desired temperature set point is
indicated. Press again to confirm the change. The display will rapidly flash the new value for a short time and then return to the
recirculating fluid temperature. Please refer to the manual for periodic maintenance.
Thermal Circulator Quick Setup
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Peristaltic Pump Quick Setup
The L/S EASY-LOAD Pump Head is compatible with most MASTERFLEX® L/S® drives with a standard tang interface. Mount the Pump Heads using the 2 long screws provided. The pump heads line up with each other by the tangs and are held together with the long thumb screws.
Be sure the pump drive is turned off. To open the pump head rotate lever to the left.
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Peristaltic Pump Quick Setup cont.
1. With the lever released, load tubing of the correct size. Center the tubing between the retainers. Make sure that the tubing is in the tubing retainer slot on each side of the pump head.
2. Rotate the lever to the right to close.
NOTE: Tubing retainers usually donot need to be readjusted whenchanging tubing of the same typeand size.
NOTE: There are a few combinations of tubing and materials that may require a further adjustment of the tubing retainers if the tubing creeps. Open pump head, move tubing retainer down one (1) notch, then close pump head. Turn pump on. If tubing creep persists, repeat procedure.
3. To set the tubing retainer to the tubing, release the retainer from the locked position by first pushing in slightly towards the body, then downward firmly against the tubing. Set retainer on both sides.
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Heart
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Water Jacket Diagram
When connecting the Water Jacketed tubing it is very important to have the flow of the water go from the bottom of the component to the top. Make sure that you remove all air bubbles from the components.
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Heart
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The simplest measurement of contractile force is made using a force transducer tied to the apex of the heart with a pulley in-between the heart and the transducer. In this system a measurable amount of force is lost in a rotational motion as the heart contracts, which some investigators compensate for with a three-point mount.
To Force Transducer
Apical Force Measurement
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The experimenter must decide whether the heart will be paced or allowed to beat spontaneously. Pacing is used to maintain a standard contractile response and metabolic demand. Spontaneous beating may permit the experimenter to measure changes in heart rate and rhythm that will occur with various drugs or manipulations.
To pace a heart, the stimulus rate must exceed the natural cardiac pacemaker rate. Often the sinoatrial node is crushed or the right atrium excised to eliminate the contribution of the primary intrinsic pacemaker. Pacing voltage is determined as a set percentage (normally 110-150%) above the voltage required to capture (pace) the heart and should not have to exceed 3-5 v, with a duration of 0.1-1 msec. Hearts may be paced using either surface or plunge electrodes inserted into the cardiac tissue by running Teflon-coated wires into needles, exposing the tips of the wires and bending the wires over the tips of the needles. The needles are then pushed into the heart and withdrawn, leaving the wire embedded in the tissue.
Should tissue damage be an issue, it is recommended that the Radnoti Pacing Electrode be used.
Pacing may also be used to induce arrhythmias in attempts to measure changes in fibrillation threshold.
Cardiac Pacing
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Heart
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(In Langendorff modes only)
A saline-filled balloon catheter inserted into the left ventricle is often used to measure isovolumetric work.
Balloons should be slightly larger than the maximum expanded volume of the ventricle to avoid effects of measuring the resistance of the balloon to stretch.
The balloon is secured to a flexible balloon catheter, which is then connected to a pressure transducer. The balloon may be inserted by passage through the left atrium or by passing the catheter through the wall of the left ventricle for pressure measurements. In this case, a one-way valve must be placed in the aortic cannula if the intraventricular pressure exceeds the perfusion pressure.
Radnoti offers a complete range of latex balloons for use in the isolated heart.
Left Ventricular Pressure
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Heart
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In the Working Heart Model, contractile function can be assessed by the initial ejection pressure at the aorta. The concomitant ability to pump against an afterload and/or reach a set ejection pressure with a preload is set by adjusting the height of the compliance loop reservoir.
Pressure-volume work is determined by the total volume of fluid ejected by the ventricle over time. In any of these cases, the experimenter should determine the appropriate amount of resting force or pressure required to maintain the heart on the ascending limb of the Starling Curve and avoid overstretching the heart muscle.
Other useful functions derived from contractile measurements include the first derivative, dP/dt, a determinant of the rate of change of developed pressure and the integral of pressure as an index of work. Heart rate can be monitored from force measurements or monitored independently with an ECG amplifier.
Afterload pressure
Working Heart Contractile Function
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The Frank-Starling law of the heart (also known as Starling's law or the Frank-Starling mechanism) states that the more the ventricle is filled with blood during diastole (end-diastolic volume), the greater the volume of ejected blood will be during the resulting systolic contraction (stroke volume). There is an optimum end-diastolic volume, after which cardiac performance declines (see below).This means that the force of contractions will increase as the heart is filled with more blood and is a direct consequence of the effect of an increasing load on a single muscle fiber. In particular, such increased load stretches the myocardium further and enhances the affinity of troponin C for calcium, hence increasing the contractile force. The force that any single muscle fiber generates is proportional to the initial sarcomere length (known as preload), and the stretch on the individual fibers is related to the end-diastolic volume of the ventricle. In the human heart, maximal force is generated with an initial sarcomere length of 2.2 micrometers, a length which is rarely exceeded in the normal heart. Initial lengths larger or smaller than this optimal value will drop the force of the muscle owing to less overlap of the thin and thick filaments for larger values and more overlap of the thin filaments for smaller values. This can be seen most dramatically in the case of a premature ventricular contraction. The premature ventricular contraction causes early emptying of the left ventricle (LV) into the aorta. Since the next ventricular contraction will come at its regular time, the filling time for the LV increases, causing an increased LV end diastolic volume. Because of the Frank-Starling law, the next ventricular contraction will be more forceful, causing the ejection of the larger than normal volume of blood, and bringing the LV end-systolic volume back to baseline.For example, during vasoconstriction the end diastolic volume increases. Increasing preload will increase stroke volume. The heart will pump what it receives. The above is true of healthy myocardium. In the failing heart with an over-dilated ventricle, cardiac performance is compromised as described by Laplace's law.
Starling Curve Defined
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LVP measurements are greatly affected by balloon size selection and pre-load based on a Starling curve.
Select the appropriate balloon size.
It is imperative that the balloon size selected not be too small for the donor heart. An indication of too small balloon size is that diastolic minimum pressure is too high in order to achieve maximum developed pressure.
Trouble ShootingLVP Max Developed Pressure and Preload (Balloon Method, Langendorff Only).
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Performing the starling curve to determine optimum preload to left ventricular balloon.
The preload on the balloon should be increased gradually while monitoring the developed pressure. An increase increment of 2mmHg followed by a review of maximum developed pressure and systolic pressure. Continue with the process until such time as an optimum developed pressure is achieved while maintaining a physiological normal systolic pressure.
Trouble ShootingLVP Max Developed Pressure and Preload (Balloon Method, Langendorff only) cont.
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Trouble ShootingThe Frank-Starling Law of the Heart (also known as Starling's law or the Frank-Starling mechanism) states that the more the
ventricle is filled with blood during diastole (end-diastolic volume), the greater the volume of ejected blood will be during the
resulting systolic contraction (stroke volume).
This means that the force of contractions will increase as the heart is filled with more blood and is a direct consequence of the
effect of an increasing load on a single muscle fiber. In particular, such increased load stretches further the myocardium and
enhances the affinity of troponin C for Calcium, hence increasing the contractile force. The force that any single muscle fiber
generates is proportional to the initial sarcomere length (known as preload), and the stretch on the individual fibers is related to the
end-diastolic volume of the ventricle. In the human heart, maximal force is generated with an initial sarcomere length of 2.2
micrometers, (a length which is rarely exceeded in the normal heart). Initial lengths larger or smaller than this optimal value will
drop the force of the muscle owing to: less overlap of the thin and thick filaments for larger values, and more overlap of the thin
filaments for smaller values. This can be seen most dramatically in the case of a premature ventricular contraction. The premature
ventricular contraction causes early emptying of the left ventricle (LV) into the aorta. Since the next ventricular contraction will
come at its regular time, the filling time for the LV increases, causing an increased LV end diastolic volume. Because of the
Frank-Starling Law, the next ventricular contraction will be more forceful, causing the ejection of the larger than normal volume of
blood, and bringing the LV end-systolic volume back to baseline. For example, during vasoconstriction the end diastolic volume
increases, increasing preload. This will increase stroke volume. The heart will pump what it receives. The above is true of healthy
myocardium. In the failing heart, the more the myocardium is dilated, the weaker it can pump, as it then reverts to Laplace's Law.
The Frank Starling Law of the Heart
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Left Ventricular End Diastolic PressureFrank Starling Curve
Left Ventricular Pressure
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Following calibration and insertion of the balloon you will want to optimize the pre-load to obtain accurate max developed pressure measurements. This is a combination of both the resting pressure (or systole) and max developed pressure diastole.
You will see a distinct pressure wave as you begin to increase pre-load to the balloon as seen in the RED trace. Gradually increasing pre-load will increase end developed pressure, as shown in the GREEN trace.
The BLUE trace indicates the approximate pre-load and max developed pressure for a 250-300gm adult rat.
The ORGANGE wave indicates that pre-load has increased too far. Depicted in the trace as an acceptable max developed pressure but an abnormally systolic or pre-load pressure. This would also be an indication of a balloon size being too small for the donor heart.
Starling Curve
Left Ventricular Pressure
Left Ventricular End Diastolic Pressure
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If the experimenter is not conversant with cardiovascular pharmacology and physiology there are a number of excellent texts for familiarization. Besides the medical physiology and pharmacology standards, there are a number of specialized texts. Pharmacologic Analysis of Drug-receptor Interaction by Terrence P. Kenakin (Raven Press, NY) is compact with practical emphasis on isolated tissues and organs in pharmacological research.
Recommended Reading
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These procedures and devices are intended for research and experimentation. All statements, technical information and recommendations herein are based on tests and sources we believe to be reliable, but the accuracy or completeness thereof is not guaranteed.
Before using, user shall determine the suitability of the product for its intended use, and user assumes all risk and liability whatsoever in connection therewith. Neither seller nor manufacturer shall be liable in tort or in contract for any loss or damage, direct, incidental, or consequential arising out of the use or the inability to use the product. No statement or recommendationcontained herein shall have any force or effect unless in an agreement signed by officers of seller and manufacturer.
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