Development of a non-depolarising normakalaemic cardioprotective preservation solution for use in orthotopic heart transplantation

Background: Since the 1970s preservation solutions for donor hearts for transplantation have relied on hypothermic depolarising potassium to arrest and protect the heart. Despite many studies investigating improvements in composition, no preservation solution has been able to provide adequate protection of the human donor heart beyond 4 – 6 hours. Suboptimal preservation and use of marginal hearts, from a diminishing donor pool, have both been linked to poor clinical outcomes. In 2004, our laboratory developed a new normokalaemic polarising cardioplegia, comprising adenosine and lignocaine (AL) for use at warmer temperatures (28 - 30°C). Subsequent studies have reported superior functional recoveries in the isolated rat heart when compared with depolarising St Thomas solution. However, the efficacy of AL cardioplegia for use at colder temperatures has not yet been investigated. The aim of this research was to develop a normokalaemic AL cardioplegic solution for use during donor heart harvest, cold storage and implantation, to possibly extend safe cold storage times to 8 hours. The performance of this normokalaemic AL preservation solution was compared with gold standard heart preservation solutions, Celsior and Histidine Tryptophan Ketoglutarate solution (Bretschneider's solution, HTK or Custodial). Methods and Experimental Design: All studies employed the isolated working rat heart following 2, 6 and 8 hours of cold storage. Primary endpoints include aortic flow, coronary flow, cardiac output, heart rate, rate pressure product, systolic and diastolic pressures. Metabolic studies include the determination of lactate levels, oxygen consumption rates, pH and troponin T release. Experimental Design: The first of the studies presented here investigated the effect of temperature on AL preservation. Functional recoveries of hearts stored in AL solution for 6 hours (4°C and 28 -30°C) were compared to those stored in Celsior. The second study was designed to evaluate a new oxygenated AL arrest/reperfusion strategy. Hearts stored in AL solution or Celsior (4°C) for 6 hours were rewarmed with oxygenated AL arrest/reperfusion strategy, and their functional recoveries were compared to hearts rewarmed in Celsior solution or Krebs Henseleit alone. The third study investigated modifications to the AL solution. Various calcium, magnesium, adenosine and lignocaine concentrations were tested over 6 hours (4°C) followed by rewarming with the oxygenated AL arrest/reperfusion strategy to determine the best solution for extended storage. The fourth study then combined higher adenosine and lignocaine concentrations (2x AL), low calcium/high magnesium (0.22 mmol/l/2.6 mmol/l) with melatonin (100 μM) and insulin (0.01 IU/ml) for storage of hearts over 8 hours followed by the oxygenated AL arrest/reperfusion strategy. Results: Following 6 hours of cold storage (4°C) AL hearts recovered 68 ± 5% of pre-arrest cardiac output compared to 47 ± 14% for hearts stored in Celsior (4°C) (p<0.05). At warmer temperatures (28 - 30°C) AL stored and flushed hearts recovered 55 ± 3% of pre-arrest cardiac output compared to Celsior hearts which failed to recover. These results showed that AL preservation was versatile at both cold and warm temperatures in the working isolated rat heart. The second study, revealed that hearts stored for 6 hours of cold storage (4°C) in AL solution and reperfused with warm oxygenated AL arrest/reperfusion strategy recovered a significantly higher cardiac output (66 ± 4% ) compared with those reperfused with warm oxygenated Krebs-Henseleit vehicle (45 ± 2% ; p<0.05). Cold Celsior hearts reperfused with warm oxygenated Celsior had 9 times higher effluent lactate values with acidosis (pH 6.5) during the last minute of rewarming compared with all groups, and this was associated with early myocardial, vascular, and electrical stunning. A surprising finding was that reperfusing cold Celsior hearts with warm oxygenated AL arrest/reperfusion strategy improved cardiac output in early reperfusion (first 10 minutes) (p<0.05) and reduced stunning. The third study showed that lowering the calcium (0.22 mM) and increasing the magnesium (2.6 mM) of the AL preservation solution for 6 hours cold storage (4°C) of isolated rat hearts followed by warming in oxygenated AL arrest/reperfusion strategy (physiological calcium and magnesium) resulted in improved functional recoveries. The cardiac output (74%) at 60 mins reperfusion was significantly better than rewarming in AL solution with low calcium/high magnesium (63%) or Krebs Henseleit solution alone (53%)(p<0.05). Interestingly this strategy also provided significantly better recoveries in early reperfusion (10 to 30 minutes), than storing hearts in AL solution with physiological levels of calcium and magnesium. Doubling the adenosine and lignocaine concentrations in the presence of low calcium/high magnesium followed by reperfusion in warm oxygenated adenosine and lignocaine with low calcium/high magnesium arrest/reperfusion strategy further improved functional recoveries. Recovery of aortic flow improved by 15% (54 to 69%, p<0.05), cardiac output by 12% (63 to 75%, p<0.05) and there were equivalent recoveries of coronary flow. In the fourth study, hearts preserved for 8 hours in AL preservation solution with low calcium/high magnesium returned 55% CO, 101% HR, and 90% to 105% systolic and diastolic pressures following 8 hours of cold storage (4°C). Combining this strategy of 2x adenosine and lignocaine concentrations with low calcium/high magnesium, melatonin (100 μmol/L) and insulin (0.01 IU/ml), for an extended arrest time of 8 hours resulted in an increased return of cardiac output (78%) with no detectable troponin T (<0.03 mg/ml). In contrast, HTK (Custodial) hearts returned 4% cardiac output, 16% heart rate, and 11% to 17% developed pressures, and troponin T was 0.13 mg/ml in effluent 5 minutes after the rewarm. Celsior hearts, despite an 86% return of heart rate, also failed to return sufficient left ventricular function (~10%) with a return of 25% cardiac output and a troponin T level of 0.24 mg/ml. Conclusions: AL cardioplegia is versatile for both cold (4°C) or warm (28 - 30°C) storage of the isolated rat heart for up to 6 hours. Hearts stored in AL generated significantly higher aortic flow, coronary flow and cardiac output than than those stored in Celsior under the same conditions. Furthermore, reperfusing and rewarming isolated rat hearts with the oxygenated AL arrest/reperfusion strategy significantly improved functional recoveries of hearts stored for 6 hours in cold adenosine and lignocaine solution and reduced stunning in the Celsior stored hearts. Clinically this finding may be of value for providing improved cardioprotection of the donor heart through the cold to warm transitions during implantation and reperfusion. Doubling the adenosine and lignocaine concentrations of the AL cardioplegia, lowering the calcium concentration to 0.22 mmol/l and increasing the magnesium concentration to 2.6 mmol/l, further improved the aortic and coronary flows following 6 hours of cold static storage; thus implying improved myocardial and coronary vascular recovery. Extended storage (8hours) of hearts in the modified AL solution augmented with melatonin and insulin, significantly improved recoveries of hearts (78% of cardiac output and no troponin T release) when compared to hearts stored in both Celsior and HTK (Custodial) which recovered less than 21% of their pre-arrest cardiac output. In conclusion, the new AL preservation solution combined with oxygenated AL arrest/reperfusion strategy may provide a new paradigm for the donor heart harvest, storage through to implantation.