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The Visible Heart® is a live beating heart functioning outside of the body under simulated physiologic conditions. Standard cardiac transplantation procedures are employed to arrest a donor heart and prepare it for reanimation. As with clinical transplantation procedures, during the time required to remove the heart and transplant it into the recipient, the heart remains inactive and is inhibited from spontaneously contracting. This is accomplished by cooling the heart in an ice slurry (hypothermia) and infusing a cold high potassium solution named cardioplegia through the coronary arteries. These same conditions or experimental modifications are employed and analyzed for effective need in our laboratory studies.
The isolated heart apparatus
is an experimental simulation of the donor recipient, providing
oxygen and metabolic substrates for the heart to survive. However,
in order to view the internal structures of the beating heart,
non-transparent blood is replaced by a clear synthetic blood-like
solution (which does not contain red blood cells.)
Since the coronary system of the heart relies on
the pressure, which enables the transport of oxygen and metabolic
substrates to the heart, initially the heart must be externally
assisted after transplantation. This is accomplished by pumping
fluid directly into the coronary system and removing fluid from
the heart’s chambers to ease the burden, a technique called
the Langendorff mode of perfusion.
Once the heart is capable of maintaining pressures and flows independently,
it is weaned (a slow transition) out of Langendorff mode and the
native flow pattern is re-established. Now the heart has fluid
movement through all four chambers and is responsible for the
work required to maintain the flow through the coronary system,
hence the name "Four Chamber Working Mode."
This preparation was reviewed and approved by the University of Minnesota Animal Use and Care Committee. All animals receive humane care in compliance with the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health. Similary, the use of the isolated human hearts has been reviewed and approved by the University of Minnesota human subjects committee.
Human hearts are obtained as generous gifts from LifeSource Upper Midwest Organ Procurement Organization, Inc, (St. Paul, Minnesota). This research is made possible due to the generous gifts of individuals whose hearts have been donated for research purposes. Their final act of generosity will enhance understanding of the inner workings of the human heart and contribute to lifesaving advances in cardiac medicine and medical device technology.
The Preparation
With continuous low flow cardioplegia perfusion
to maintain cardiac arrest, the major vessels of the swine, canine,
or human heart are cannulated while the excised heart remains in
a buffer slurry. The inferior vena cava, pulmonary artery, right
and left superior pulmonary veins, and aorta are fitted with clear
tygon tubing and then connected to the isolated heart apparatus.
In addition, the superior vena cava and the innominate artery
of the aorta are cannulated to serve as camera ports for filming
right and left side anatomy, respectively. After approximately
1-3 hours of cardioplegia perfusion, the heart is reanimated with
the flow of an oxygenated clear crystalloid perfusate. The temperature
of the perfusate is slowly increased over a 15-30 min. period
until cardiac temperatures are raised to and maintained at approximately 37.5°C.
Initially, the heart is perfused using the method of Langendorff
as this provides the myocardium with the oxygen and metabolites
that have been depleted during the ischemic period. Once the heart
is warmed, spontaneous electrical activity returns. If normal
atrioventricular electrical activity (i.e. sinus rhythm) is not
present upon reperfusion, a 10-34 Joule defibrillation shock is delivered. This procedure is repeated until normal sinus rhythm
is observed. After 10 minutes of observed sinus rhythm with the
absence of severe arrhythmias, the apparatus can be modified so
as to allow the heart to resume its intrinsic functional activity
in a 4 chamber working mode.
Langendorff Constant Pressure Flow
In 1895, Oscar Langendorff was the first to produce an isolated mammalian heart with full contractile activity. Since that first experiment, the method of Langendorff has been a pillar in cardiac research leading to major advancements in the field of cardiology. The basic goal of the Langendorff method is to provide an isolated heart with oxygen and metabolites via a single cannula inserted into the ascending aorta. Oxygenated blood or a perfusate is pumped down the aorta towards the heart by means of an external pump. This constant 'retrograde' perfusion of the aorta keeps the aortic valve closed and allows for fluid flow into the coronary arteries during the diastolic period, just as it flows in the normal cardiac cycle. Fluid (buffer) movement continues through the coronary system (left and right main coronaries -> arterial branches -> arteriole -> capillaries -> venilles -> coronary veins) and eventually exits via the coronary sinus in the right atrium. Approximate coronary flows required for a large mammalian heart range between 0.5 – 1.5 Liters/min.
Throughout this procedure, the left chambers of the heart remain filled and the ventricles contract, but without fluid exchange (no flow). Since the force of contraction is proportional to the pressure inside the ventricles (Frank-Starling principle), many researchers have attempted to pressurize the left ventricle to create a more physiological contraction. For example, inflated balloons have been placed in the ventricles to increase the ventricular pressure and the force of contraction. Similarly, pressures have been created by filling the ventricles with fluid and using an atrioventricular clamp to prevent valvular regurgitation, as is done in our laboratory.
Initially, the Visible Heart® preparation relies
on constant pressure Langendorff perfusion to supply the myocardium
with adequate oxygen and metabolites that may have been depleted
during explantation. However, once normal sinus rhythm has been
sustained without the presence of major arrhythmias, the heart
can be transitioned into 4 chamber working mode and Langendorff
perfusion stopped or re-established as desired.
Four Chamber Working Mode
Four chamber working mode perfusion differs from the Langendorff mode in a number of aspects. First, there is two-way flow through the aorta whereas only retrograde flow existed during Langendorff mode. Second, all 4 chambers of the heart are filled with changing volumes and the natural flow through the heart is preserved; the atrial flow empties into the ventricle during diastole where it is then ejected from the ventricle during systole. Third, the aortic valve opens and closes as the heart contracts. Finally, the flow into the coronaries is determined by the contraction/performance of the heart itself and not by an external pump, as in the Langendorff mode.
Working mode has also been called physiological perfusion because its objective is to best simulate or recreate the in vivo perfusion and flows through the heart. The preload and afterload provided by the isolated heart apparatus are the primary determinants of flow through the 4 heart chambers and the coronary system. For example, a rise in the preload will increase atrial and ventricular filling which translates into greater contractility (Frank-Starling principle). However, overdistension of the ventricle will increase the oxygen demand and have a negative effect on contractility. Afterload is characteristic of the pressure work the ventricle must do to overcome the impedance to ejection created by the diastolic pressure in the aorta. A reduction of afterload will increase cardiac output but extreme hypotension will decrease coronary flow and impair contractility. If afterload is increased, the stroke volume and ejection fraction will decline. Therefore, by adjusting the preloads and afterloads various cardiac states can be simulated in the Visible Heart® apparatus of both the normal or impaired (diseased) heart.
The Apparatus
The isolated heart apparatus serves the dual role of allowing for operation in both the Langendorff and working modes. Constant pressure Langendorff perfusion of the coronaries can easily be modified to allow the heart to self-perfuse by simply inhibiting the flow of buffer through the “Langendorff Bypass”.
During Langendorff mode perfusion, the left side afterload is held constant and the flow through the coronaries is determined by dilation or constriction of the coronary arteries. The right and left side holding chambers are closed so there is no flow into the heart by way of the inferior vena cava or pulmonary vein. After the buffer exits the coronary system by way of the coronary sinus located in the right atrium, the buffer flow proceeds into the right ventricle and is ejected out of the pulmonary artery.
During working mode the flow through the heart is normally determined by the intrinsic heart rate and the contractility of the heart. The intrinsic heart rate can be modified by altering the temperature of the buffer or via the addition of pharmacological agents. By providing near physiological preload and afterload pressures, the apparatus can allow a heart to function in an almost a one-to-one re-creation of the in situ heart (see Results) for experimentally useful periods of time (up to several hours).
Cardioplegia
Typically, St. Thomas' Hospital cardioplegia solution is used to arrest the heart within seconds of the solution being introduced into the coronary blood stream. The cardioplegia consists of a potassium concentration four times the normal concentration found in the blood (17 mmoles/L). Consequently, the myocardial cells in the conduction pathway are depolarized by the influx of potassium ions. The cells remain at a neutral polarity due to the potassium imbalance and repolarization will not occur until the extracellular potassium is removed. Not only does cardioplegia prevent normal conduction from the SA node to the rest of the myocardium, but it also prevents the occurrence of ectopic beats, which may originate in any cell of the treated myocardium.
During the explantation procedure no oxygen is delivered to the heart via the coronary system (this state is known as global ischemia). Thus, any cardiac activity that occurs while the heart is ischemic will result in depletion of vital energy needed for the cell to survive. Ischemia has also been speculated to cause an increase in oxygen free radical formation and altered capillary membrane permeability. By continuing the flow of cardioplegia during the entire explantation procedure and inhibiting any cardiac activity, the amount of ischemic damage to the myocardium is sought to be minimized.
The effects of various methods to improve ischemic
tolerance are currently being investigated using the Visible Heart®
preparation; e.g. by changing the osmolarity, pH, and/or composition
of the cardioplegia solution, the in vitro performance
of the heart can be improved and the life of the preparation been
prolonged. Additionally, preconditioning of the heart with various
therapeutic agents is another pharmacological approach currently
being pursued in our lab to improve post-ischemic recovery of
the heart. (see references)
Crystalloid versus Colloid Perfusates
One of the most important attributes of the modified Krebs-Henseleit buffer used in this preparation is its transparency. This crystalloid solution is essentially a balanced salt solution with glucose added as an energy substrate; it may or may not contain proteins. Potential disadvantages of a crystalloid perfusate are its decreased ability to carry oxygen over extended lengths of tubing and its low viscosity. Furthermore, the addition or removal of small amounts of salt can cause large changes in the osmolarity, an important determinate of water weight gain by the heart tissue (edema). A solution containing colloids such as cells, proteins, and synthetic macromolecules minimizes changes in osmolarity and increases the viscosity of the solution to more physiological levels (i.e. that of blood). However, these colloid additives may compromise the transparency of the solution and if they cross the endothelial membrane of the blood vessels, edema could be accelerated. Our lab is actively investigating such modification of our perfusates.
References:
Langendorff, O. Untersuchungen am überlebenden Säugethierherzen. Pflügers Arch. ges. Physiol. 61, 291 (1895)Döring, HJ,
Dehnert H. The Isolated Perfused Warm-Blooded Heart according to Langendorff. Methods in Experimental Physiology and Pharmacology: Biological Measurement Techniques V. Biomesstechnik-Verlag March GmbH, West Germany. 1988.
Chinchoy
E, Soule CL, Houlton AJ, Gallagher WJ, Hjelle MA, Laske TG, Morissette
J, Iaizzo PA. Isolated four-chamber working swine heart model.
Ann Thorac Surg. 2000 Nov;70(5):1607-14
Hill AJ, Coles JA, Sigg DC, Laske TG, Iaizzo PA. Images of the Human Coronary Sinus Ostium Obtained from Isolated Working Hearts. Ann Thorac Surg. 2003;76:2108
Hill AJ, Coles JA, Sigg DC, Laske TG, Soule CL, Gallagher WJ, Iaizzo PA. In Vitro Studies of Human Hearts. Ann Thorac Surg. V. 79, pp. 168-77, 2005.
Sigg DC, Iaizzo PA. Malignant hyperthermia phenotype: hypotension induced
by succinylcholine in susceptible swine. Anesthesiology 2000 Jun;92(6):1777-88.
Sigg DC, Coles JA, Gallagher WJ, Oeltgen PR, Iaizzo PA. Opioid preconditioning: Myocardial Function and energy metabolism. Ann Thorac Surg. 2001;72:1576-82.
Hill AJ, Coles JA, Sigg DC, Laske TG, Iaizzo PA. Cardiac Anatomy: Fixed and Functional Comparisons of Human, Porcine, Canine, and Ovine Hearts. FASEB 2002;16(4):(Abstract 780.14).
Hill AJ, Coles JA, Sigg DC, Laske TG, Iaizzo PA. In Vitro Studies of Human Hearts: A Four-Chamber Working Isolated Heart Model. FASEB 2002;16(4):A383(Abstract 328.4).
Laske TG, Skadsberg ND, Iaizzo PA. Comparative In Vivo and Ex Vivo Pacing and Sensing Performance Study Using Isolated Four-Chamber Working Swine Heart Model. IEEE Transactions, Oct 2002; pp.244 [A.6.1.4-3].
Skadsberg ND, Laske TG, Hill AJ, Iaizzo, PA, Correlation of cardiac activation to anatomy in an isolated heart model using non-contact mapping. FASEB April 2003; The FASEB Journal, V. 17(4) [Pt. 1], p. A117 [A 98.7].
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