Biochemistry
 
 Prolonged Preservation of the Heart Prior to Transplantation
 
 
 
         Picture this.  A man is involved in a severe car crash in 
 Florida which has left him brain-dead with no hope for any 
 kind of recovery.  The majority of his vital organs are 
 still functional and the man has designated that his organs 
 be donated to a needy person upon his untimely death.  
 Meanwhile, upon checking with the donor registry board, it 
 is discovered that the best match for receiving the heart of 
 the Florida man is a male in Oregon who is in desperate need 
 of a heart transplant.  Without the transplant, the man will 
 most certainly die within 48 hours.  The second man's 
 tissues match up perfectly with the brain-dead man's in 
 Florida.  This seems like an excellent opportunity for a 
 heart transplant.  However, a transplant is currently not a 
 viable option for the Oregon man since he is separated by 
 such a vast geographic distance from the organ.  Scientists 
 and doctors are currently only able to keep a donor heart 
 viable for four hours before the tissues become irreversibly 
 damaged.  Because of this preservation restriction, the 
 donor heart is ultimately given to someone whose tissues do 
 not match up as well, so there is a greatly increased chance 
 for rejection of the organ by the recipient.  As far as the 
 man in Oregon goes, he will probably not receive a donor 
 heart before his own expires.
 
         Currently, when a heart is being prepared for 
 transplantation, it is simply submerged in an isotonic 
 saline ice bath in an attempt to stop all metabolic activity 
 of that heart.  This cold submersion technique is adequate 
 for only four hours.  However, if the heart is perfused with 
 the proper media, it can remain viable for up to 24 hours.  
 The technique of perfusion is based on intrinsically simple 
 principles.  What occurs is a physician carefully excises 
 the heart from the donor.  He then accurately trims the 
 vessels of the heart so they can be easily attached to the 
 perfusion apparatus.  After trimming, a cannula is inserted 
 into the superior vena cava.  Through this cannula, the 
 preservation media can be pumped in.
 
         What if this scenario were different?  What if doctors were 
 able to preserve the donor heart and keep it viable outside 
 the body for up to 24 hours instead of only four hours?  If 
 this were possible, the heart in Florida could have been 
 transported across the country to Oregon where the perfect 
 recipient waited.  The biochemical composition of the 
 preservation media for hearts during the transplant delay is 
 drastically important for prolonging the viability of the 
 organ.  If a media can be developed that could preserve the 
 heart for longer periods of time, many lives could be saved 
 as a result.
 
         Another benefit of this increase in time is that it would 
 allow doctors the time to better prepare themselves for the 
 lengthy operation.  The accidents that render people 
 brain-dead often occur at night or in the early morning.  
 Presently, as soon as a donor organ becomes available, 
 doctors must immediately go to work at transplanting it.  
 This extremely intricate and intense operation takes a long 
 time to complete.  If the transplanting doctor is exhausted 
 from working a long day, the increase in duration would 
 allow him enough time to get some much needed rest so he can 
 perform the operation under the best possible circumstances.
 
         Experiments have been conducted that studied the effects of 
 preserving excised hearts by adding several compounds to the 
 media in which the organ is being stored.  The most 
 successful of these compounds are pyruvate and a pyruvate 
 containing compound known as 
 perfluoroperhydrophenanthrene-egg yolk phospholipid 
 (APE-LM).  It was determined that adding pyruvate to the 
 media improved postpreservation cardiac function while 
 adding glucose had little or no effect.  To test the 
 function of these two intermediates, rabbit hearts were 
 excised and preserved for an average of 24.5 1 0.2 hours on 
 a preservation apparatus before they were transplanted back 
 into a recipient rabbit.  While attached to the preservation 
 apparatus, samples of the media output of the heart were 
 taken every 2 hours and were assayed for their content.  If 
 the compound in the media showed up in large amounts in the 
 assay, it could be concluded that the compound was not 
 metabolized by the heart.  If little or none of the compound 
 placed in the media appeared in the assay, it could be 
 concluded that compound was used up by the heart metabolism.
 
         The hearts that were given pyruvate in their media 
 completely consumed the available substrate and were able to 
 function at a nearly normal capacity once they were 
 transplanted.  Correspondingly, hearts that were preserved 
 in a media that lacked pyruvate had a significantly lower 
 rate of contractile function once they were transplanted.  
 The superior preservation of the hearts with pyruvate most 
 likely resulted from the hearts use of pyruvate through the 
 citric acid cycle for the production of energy through 
 direct ATP synthesis (from the reaction of succinyl-CoA to 
 succinate via the enzyme succinyl CoA synthetase) as well 
 as through the production of NADH + H+ for use in the 
 electron transport chain to produce energy.  
 
                 After providing a preservation media that contained 
 pyruvate, a better recovery of the heart tissue occurred.  
 Most of the pyruvate consumed during preservation was 
 probably oxidized by the myocardium in the citric acid 
 cycle.  Only a small amount of excess lactate was detected 
 by the assays of the preservation media discharged by the 
 heart.  The lactate represented only 15% of the pyruvate 
 consumed.  If the major metabolic route taken by pyruvate 
 during preservation had been to form lactate dehydrogenase 
 for regeneration of NAD+ for continued anaerobic glycolysis, 
 rather than by the aerobic citric acid cycle (pyruvate 
 oxidation), then a higher ratio of excess lactate produced 
 to pyruvate consumed would have been observed.
 
         Hearts given a glucose substrate did not transport or 
 consume that substrate, even when it was provided as the 
 sole exogenous substrate.  It might be expected that glucose 
 would be used up in a manner similar to that of pyruvate.  
 This expectation is because glucose is a precursor to 
 pyruvate via the glycolytic pathway however, this was not 
 the case.  It was theorized this lack of glucose use may 
 have been due to the fact that the hormone insulin was not 
 present in the media.  Without insulin, one may think the 
 tissues of the heart would be unable to adequately take 
 glucose into their tissues in any measurable amount, but 
 this is not the case either.  It is known that hearts 
 working under physiologic conditions do use glucose in the 
 absence of insulin, but glucose consumption in that 
 situation is directly related to the performance of work by 
 the heart, not the presence of insulin.  
 
         To further test the effects of the addition of insulin to 
 the glucose media, experiments were done in which the 
 hormone was included in the heart preservation media5-7.  
 Data from those studies does not provide evidence that the 
 hormone is essential to insure glucose use or to maintain 
 the metabolic status of the heart or to improve cardiac 
 recovery.  In a hypothermic (80C) setting, insulin did not 
 exert a noticeable benefit to metabolism beyond that 
 provided by oxygen and glucose.  This hypothermic setting is 
 analogous to the setting an actual heart would be in during 
 transportation before transplant.  
 
         Another study was done to determine whether the compound 
 perfluoroperhydrophenanthrene-egg yolk phospholipid, 
 (APE-LM) was an effective media for long-term hypothermic 
 heart preservation3.  Two main factors make APE-LM an 
 effective preservation media.  (1) It contains a lipid 
 emulsifier which enables it to solubilize lipids.  From this 
 breakdown of lipids, ATP can be produced.  (2) APE-LM 
 contains large amounts of pyruvate.  As discussed earlier, 
 an abundance of energy is produced via the oxidation of 
 pyruvate through the citric acid cycle.  
         
         APE-LM-preserved hearts consumed a significantly higher 
 amount of oxygen than hearts preserved with other media.  
 The higher oxygen and pyruvate consumption in these hearts 
 indicated that the hearts had a greater metabolic oxidative 
 activity during preservation than the other hearts.  The 
 higher oxidative activity may have been reflective of 
 greater tissue perfusion, especially in the coronary beds, 
 and thereby perfusion of oxygen to a greater percentage of 
 myocardial cells.  Another factor contributing to the 
 effectiveness of APE-LM as a transplantation media is its 
 biologically compatible lipid emulsifier, which consists 
 primarily of phospholipids and cholesterol.  The lipid 
 provides a favorable environment for myocardial membranes 
 and may prevent perfusion-related depletion of lipids from 
 cardiac membranes.  The cholesterol contains a bulky steroid 
 nucleus with a hydroxyl group at one end and a flexible 
 hydrocarbon tail at the other end.  The hydrocarbon tail of 
 the cholesterol is located in the non polar core of the 
 membrane bilayer.  The hydroxyl group of cholesterol 
 hydrogen-bonds to a carbonyl oxygen atom of a phospholipid 
 head group.  Through this structure, cholesterol prevents 
 the crystallization of fatty acyl chains by fitting between 
 them.  Thus, cholesterol moderates the fluidity of 
 membranes.8
 
         The reason there are currently such strict limits on the 
 amount of time a heart can remain viable out of the body is 
 because there must be a source of energy for the heart 
 tissue if it is to stay alive.  Once the supply of energy 
 runs out, the tissue suffers irreversible damage and dies.  
 Therefore, this tissue cannot be used for transplantation.  
 If hypothermic hearts are not given exogenous substrates 
 that they can transport and consume, like pyruvate, then 
 they must rely on glycogen or lipid stores for energy 
 metabolism.  The length of time that the heart can be 
 preserved in vitro is thus related to the length of time 
 before these stores become too low to maintain the required 
 energy production needs of the organ.  It is also possible 
 that the tissue stores of ATP and phosphocreatine are 
 critical factors.  It is known that the amount of ATP in 
 heart muscle tissues is sufficient to sustain contractile 
 activity of the muscle for less than one second.  This is 
 why phosphocreatine is so important.  Vertebrate muscle 
 tissue contains a reservoir of high-potential phosphoryl 
 groups in the form of phosphocreatine.  Phosphocreatine can 
 transfer its phosphoryl group to ATP according to the 
 following reversible reaction:
 
                 phosphocreatine  +  ADP  +  H+   9   ATP  +  creatine
 
 Phosphocreatine is able to maintain a high concentration of 
 ATP during periods of muscular contraction.  Therefore, if 
 no other energy producing processes are available for the 
 excised heart, it will only remain viable until its 
 phosphocreatine stores run out.
 
 
         A major obstacle that must be overcome in order for heart 
 transplants to be successful, is the typically prolonged 
 delay involved in getting the organ from donor to recipient.  
 The biochemical composition of the preservation media for 
 hearts during the transplant and transportation delays are 
 extremely important for prolonging the viability of the 
 organ.  It has been discovered that adding pyruvate, or 
 pyruvate containing compounds like APE-LM, to a preservation 
 medium greatly improves post-preservation cardiac function 
 of the heart.  As was discussed, the pyruvate is able to 
 enter the citric acid cycle and produce sufficient amounts 
 of energy to sustain the heart after it has been excised 
 until it is transplanted.
         
         Increasing the amount of time a heart can remain alive 
 outside of the body prior to transplantation from the 
 current four hours to 24 hours has many desirable benefits.  
 As discussed earlier, this increase in time would allow 
 doctors the ability to better match the tissues of the donor 
 with those of the recipient.  Organ rejection by recipients 
 occurs frequently because their tissues do not suitably 
 match those of the donors.  The increase in viability time 
 would also allow plenty of opportunity for the organ to be 
 transported to the needy person, even if it must go across 
 the country.