Prolonged Hypothermic Storage Research Articles

Pretreatment of brain dead rabbits with pinacidil before prolonged cold-storage with an extracellular solution alters aortic endothelial function

Objective: Endothelial injury occurs during heart transplantation and contributes to the development of cardiac allograft vasculopathy. We have evaluated in a brain death model in the rabbit whether pre-treatment with the potassium channel opener (PCO) pinacidil before prolonged hypothermic storage with an extracellular solution would improve vascular endothelial recovery. Methods Rabbits were randomized into 4 experimental groups ( n = 8 per group). In the control group (CTRL), abdominal aortic rings were assessed immediately after 90 minutes of anesthesia. In the brain death group (BD), aortic rings were assessed immediately after 90 minutes of brain death. In the STH group, aortic rings taken from brain dead rabbits were stored for 24 hours at 4°C with the extracellular preservation solution of St. Thomas Hospital (STH) before assessment. In the STH + PCO group, the potassium channel opener pinacidil, 1 mg/kg, was administered intravenously to brain dead rabbits 10 minutes before explantation. Aortic rings were then stored for 24 hours at 4°C with the STH solution before evaluation. Brain death was induced by rapid inflation of a sub-durally placed balloon and validated by clinical and electroencephalographic data. Concentration-response curves to acetylcholine (ACH, 10 -9 to 10 -4 mol/liter) and nitroglycerin (NGL, 10 -9 to 10 -5 mol/liter) were constructed in phenylephrinepre-contracted rings. Results ACH evoked a similar concentration-dependent relaxation in the CTRL (E max: 95.8 ± 2.9%; EC 50: -6.86 ± 0.13 log M) and BD groups (E max: 90.8 ± 3.8%; EC 50: -6.75 ± 0.15 log M). The concentration-relaxation curve was shifted rightward in the STH group (E max: 76.7 ± 7.1%; EC 50: -6.75 ± 0.16 log M) in comparison with the CTRL and BD groups, but there were no significant differences in either E max or EC 50 values. After pinacidil pre-treatment, there was a further significant shift to the right of the concentration-relaxation curve to ACH (E max: 77.4 ± 5.0%; EC 50: -6.14 ± 0.19 log M, p < 0.05 vs CTRL, BD and STH). There were no significant differences between groups in the concentration-relaxation curves to NGL in endothelium-intact and endothelium-denuded vascular rings (either E max or EC 50). Conclusion Pre-treatment of brain dead rabbits with pinacidil before prolonged cold-storage with STH solution significantly impaired endothelium-dependent vasorelaxation in comparison to storage with STH solution. The role of PCO pre-treatment in the context of cardiac transplantation needs to be reconsidered.

Prolonged Hypothermic Cardiac Storage with Histidine-tryptophan-ketoglutarate Solution-Comparison with Glucose-insulin-potassium and University of Wisconsin Solutions

95 This study was designed to test the efficacy of histidine-tryptophan-ketoglutarate (HTK) solution after prolonged cold storage as compared with the conventional glucose-insulin-potassium (GIK) and University of Wisconsin (UW) solutions. Methods. (1). The hearts isolated from male Wistar rats were divided into three groups (n=6 per group) according to each preservation solution used. The hearts were arrested and stored in each solution for 6, 8, and 12 hrs at 4 °C. After storage, recovery of cardiac function and myocardial adenylate and water contents were evaluated. (2). Mechanical function of the epicardial coronary arteries (left main trunks) were compared between UW and HTK solutions. Coronary vascular responsiveness were examined at four predetermined time points: prior to flush and following flush, storage, and reperfusion. Results. (1). Cardiac function, which was sufficiently maintained in the HTK group up till 8 hrs of storage decreased below 50% following 8 hrs of storage in the UW and GIK groups. Although the myocardial ATP/ADP ratio in the HTK group was maintained above 50% up till 8 hrs of storage, those ratios in the GIK and UW groups declined to 15%. The values of myocardial energy charge in the HTK group were maintained above 60% up till 8 hrs of storage, however, those values in the UW and GIK groups declined to below 50% following 6 hrs of storage. The water contents following 12 hrs of storage were lower in the HTK and UW groups than in the GIK group. (2). Following reperfusion, the maximal relaxation response to acetylcholine in the HTK group was higher than in the UW group (p<0.05), and the endothelium-independent relaxation response to sodium nitroprusside in the HTK group was also well preserved. Discussion and Conclusion. In comparison with UW and GIK solutions, HTK solution, which sustains myocardial energy level and coronary artery function during preservation may lead to a better recovery of cardiac function; however, successful cold storage of the heart is highly energy-dependent, and a dramatic breakdown of myocardial energy level, which causes a crucial decline in the cardiac function, occurs between 8 and 12 hr of storage.

ATP-sensitive potassium channel activation mimics the protective effect of ischaemic preconditioning in the rat isolated working heart after prolonged hypothermic storage.

1. Ischaemic preconditioning (IP) can significantly reduce the extent of infarct size, contractile dysfunction and necrosis in hearts from a number of animal species. Activation of ATP-sensitive potassium channels has been implicated in this process. The aims of the present study were to determine the extent to which IP preserves haemodynamic function in the rat isolated working heart model after prolonged hypothermic storage and to examine the involvement of activation of potassium channels in this process. 2. Hearts from Wistar rats were perfused on a Langendorff apparatus. After stabilization in working mode, baseline measurements of heart rate, aortic flow, coronary flow and cardiac output were performed. Hearts were randomized to one of six treatment groups: (i) untreated control; (ii) IP; (iii) 3 min perfusion with 200 mumol/L pinacidil; (iv) pinacidil vehicle; (v) 3 min perfusion with 10 mumol/L glibenclamide before IP; and (vi) 3 min perfusion with glibenclamide then pinacidil. Hearts were stored in an extracellular-based preservation solution for 6 or 12 h at 2-3 degrees C, remounted on the perfusion apparatus, stabilized as before and then haemodynamic measurements were repeated, after which time heart water contents were determined. 3. Recovery of haemodynamic function was markedly enhanced in the IP and pinacidil-treated groups compared with untreated and vehicle controls. These beneficial effects were completely blocked by glibenclamide. These results suggest that strategies for activating potassium channels in donor hearts may protect organs during hypothermic storage prior to transplantation.

Cardioprotection by Activation of NO/cGMP Pathway After Cardioplegic Arrest and 8-Hour Storage

Background. We determined whether activation of the nitric oxide/cyclic guanosine monophosphate pathway by sodium nitroprusside (SNP) protects hearts subjected to cardioplegic arrest and prolonged hypothermic storage. Methods. Isolated rat hearts arrested with St. Thomas’ II cardioplegia and stored at 3° ± 1°C for 8 hours were reperfused at 37°C in Langendorff (10 minutes) and working (60 minutes) modes. Results. During reperfusion, left ventricular work was depressed in stored hearts relative to fresh hearts. When present during arrest, storage, and both reperfusion phases, SNP (200 μmol/L) improved work to values close to those in fresh hearts. When added only during the 10-minute period of Langendorff reperfusion, SNP also improved the subsequent recovery of work. This effect was antagonized by the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Poststorage coronary perfusion was not increased by SNP. Conclusions. The ability of SNP to enhance recovery independent of changes in coronary perfusion and in an ODQ–sensitive manner suggests that SNP–induced protection is due to activation of the myocardial nitric oxide/cyclic guanisine monophosphate pathway. These results suggest that supplementing cardioplegic solutions with SNP, administering SNP during early reperfusion, or both may offer additional means to improve donor heart preservation.

Enhanced preservation of the rat heart after prolonged hypothermic storage with the 21-aminosteroid compound U74500A

Background. The formation of reactive oxygen species has been postulated to play a role in the initiation of tissue damage associated with ischaemia/reperfusion injury in donor hearts after hypothermic preservation. The aim of this study was to determine whether addition of U74500A to an aspartate-enriched, extracellular-based cardioplegic solution enhanced function of donor rat hearts after prolonged hypothermic storage. Methods: Excised hearts were ligated to an aortic cannula and immediately perfused retrogradely with oxygenated Krebs' solution. This preparation was then converted from Langendorff to working mode. After a 1-min stabilisation period, baseline measurements of heart rate, aortic flow, coronary flow and cardiac output were performed. Oxygenated cardioplegic solution, with or without U74500A (30 μM), was infused into the coronary circulation. Hearts were stored in the same solutions for 6 or 12 hours at 2–3°C. The hearts were reperfused and haemodynamic measurements were repeated. Water content of the hearts was determined. Results and Conclusions: Addition of lazaroid significantly improved all haemodynamic parameters measured after 12 hours storage and aortic flow at 6 hours storage compared with the untreated and vehicle control groups. The presence of the lazaroid, U74500A, was associated with significantly better preservation of coronary flow and cardiac function in the isolated rat heart after 6 or 12 hours of hypothermic storage. This suggests a novel use for this family of compounds in the transplantation context.

Hypothermic storage of sheep embryos with antifreeze proteins: Development in vitro and in vivo

Antifreeze proteins (AFPs) non-colligatively lower the freezing point of aqueous solutions, block membrane ion channels and thereby confer a degree of protection during cooling. Ovine embryos following prolonged hypothermic storage were used to determine 1) the type and concentration of a group of AFPs that can confer hypothermic tolerance, 2) the storage temperature, 3) the cooling rate, and 4) the in vitro and in vivo viability. In Experiment 1, Grade 1 and 2 embryos produced following superovulation were either cultured fresh (control) or stored at 4 °C for 4 d in media containing protein from 1 of 3 sources: Winter Flounder (WF; AFP Type 1); Ocean Pout (OP; AFP Type 3) at a concentration of 1 or 10 mg/ml; or bovine serum albumen (BSA) at 4 mg/ml in phosphate buffered saline (PBS). Following 72 h of culture, the viability rates were not different between controls ( 18 21 ); BSA ( 9 15 ); WF at 1 mg/ml ( 14 15 ); WF at 10 mg/ml ( 13 15 ) or OP at I mg/n-d ( 15 21 ), but were decreased (P < 0.05) in embryos stored in OP at 1 0 mg/ml (I 1 20 ). Pooled data showed higher (P < 0.05) viability rates for WF ( 27 30 ) than for OP ( 26 41 ) or BSA ( 9 15 ). There was no effect of protein source on hatching rates, but mean hatched diameters of embryos were lower (P < 0.05) following storage in BSA. In Experiment 2, Grade I to 3 embryos were either cultured fresh or stored for 4 d at 0 ° or 4 °C in 4 mg/n-d BSA or 1 mg/ml WF. Embryos stored in WF at 4 °C (WF/4 °C) had comparable hatching rates ( 8 12 ) to that of controls ( 10 10 ), but embryos in the other treatments (WF 0 °C, 5 11 , BSA 4 °C, 6 11 and BSA 0 °C, 3 10 ) had significantly lower hatching rates (P < 0.01) compared with controls. Hatched diameters were comparable between controls and embryos stored in WF 4 °C, but embryos stored in WF 0 °C and BSA at both temperatures had smaller diameters (P < 0.05). In Experiment 3, Grade 1 to 3 embryos were either transferred fresh or were stored for 4 d at 4 °C in 4 mg/ml BSA or 1 mg/ml WF at different cooling rates (T1, BSA > 2 °C/min; T2, WF > 2 °C/min and T3, WF < 1 °C/min) prior to transfer. There were no differences in the number of ewes pregnant (T1, 10 1 1; T2, 6 10 and T3, 8 10 ) or in the number of viable fetuses recovered per treatment (T1, 14 25 ; T2, 10 1 4 and T3, 15 2 1) to indicate a negative effect of cooling rate or protein on embryo survival. In conclusion, ovine embryos can be stored in WF or BSA at 4 °C for 4 d, yielding similar pregnancy and embryo survival rates as fresh embryos following transfer to recipient ewes.

Editorial: Sodium-hydrogen exchange inhibition—superior cardioprotective strategy

Editorial: Sodium-hydrogen exchange inhibition—superior cardioprotective strategy

The Beneficial Effects of Heat-Shock for Prolonged Hypothermic Storage

Heat-shock has been reported to induce tolerance for subsequent ischemia. We wished to determine whether thermal stress is protective for hypothermic storage. Three groups of Sprague-Dawley rats were studied (n= 8 per group). Control animals received routine care (CONT) while anaesthetized rats were either warmed to 42°C for 20 min (HEAT) or maintained at room temperature (SHAM). Twenty-four hours later, hearts were mounted on a Langendorff apparatus with an intraventricular balloon. Hearts were then flushed and stored in UW solution for 8 hr at 0°C and reperfused for 45 min. Data are reported as a percentage of the prestorage results or as the absolute value (mean ± SD). Recovery of developed pressure was significantly greater (P< 0.05) in the heat-shocked animals (79.5 ± 10.2%) than in the SHAM (63.6 ± 17.2%) or CONT groups (59.0 ± 10.8%). Coronary flow was similarly enhanced (P< 0.05) in the HEAT group (86.8 ± 5.5%) vs the CONT (77.0 ± 12.3%) or SHAM hearts (74.5 ± 10.2%). Diastolic compliance as assessed by evaluation of the end-diastolic pressure-volume curves was reduced in all groups (P< 0.0001) but not different among groups. Cardiac creatine kinase release during reperfusion was greater in the CONT and SHAM groups (CONT: 726.9 ± 297.8 IU/g; SHAM: 548.9 ± 420.9 IU/g) than in the heat-shocked rodents (282.3 ± 175.5 IU/g,P< 0.05 HEAT vs CONT). Cardiac biopsies were performed sequentially in separate animals (n= 6 per group). Tissue levels of ATP and total adenine nucleotides were greater in the heat-shocked or SHAM hearts following reperfusion compared with controls (P< 0.05). Thermal stress provides additional protection for prolonged hypothermic storage.

Improved cardiac function after prolonged hypothermic ischemia with the Na +/H + exchange inhibitor HOE 694

Na+/H+ exchange represents an important mechanism for pH regulation in the cardiac cell that, however, may paradoxically mediate tissue damage in the reperfused myocardium. We investigated whether inhibition of the exchanger can protect the heart against damage after prolonged hypothermic storage with the use of the selective inhibitor 3-methylsulfonyl-4-piperidinobenzoyl-guanidine methanesulfonate (HOE 694). After equilibration, isolated rabbit hearts were arrested with a 3 minute infusion of modified St. Thomas' cardioplegic solution and subsequently maintained in ischemic arrest at 4 degrees C for 12 hours before reperfusion at 37 degrees C for 60 minutes. Left ventricular function and creatine kinase release were measured at intervals throughout reperfusion. High-energy phosphate and adenine nucleotide content were determined in hearts before cardioplegia, at the end of the 12-hour storage period, and at the end of reperfusion. HOE 694 (1 mumol/L) was administered either with cardioplegia and throughout reperfusion (study 1) or selectively with either cardioplegia or reperfusion only (study 2). In study 1, systolic function in untreated hearts recovered to less than 40% of preischemic values and was associated with a greater than 1,000% percent sustained elevation in left ventricular end-diastolic pressure. In contrast, systolic recovery in HOE 694-treated hearts was significantly accelerated and improved to approximately 80%, whereas left ventricular end-diastolic pressure increased to only 300% of baseline. Significant protection also occurred in those hearts in which HOE 694 was administered only at reperfusion while the drug was less effective if given only during cardioplegia. Creatine kinase release was not significantly affected except in study 2, where it was significantly lower after 60 minutes of reperfusion in hearts where HOE 694 was added at the time of reperfusion. Tissue metabolite content was not affected by drug treatment. This study shows a marked protective effect of the Na+/H+ exchange inhibitor HOE 694 in rabbit hearts subjected to 12 hours of hypothermic ischemia and strongly suggests that antiport inhibitors could play an effective role in myocardial preservation.

Cardioprotective effects of the lazaroid U74389G following cold preservation and transplantation of rat hearts.

Limited recovery of contractile function and loss of coronary reactivity have been observed following prolonged hypothermic storage and transplantation of the heart. Since lipid peroxidation has a significant role in these deficits, we investigated the cardioprotective effects of a 21-aminosteroid. U74389G, 3 mg/kg i.v., was given daily for 2 consecutive days to donor Lewis rats before the hearts were harvested and to recipient Lewis rats for 3 consecutive days after heart transplantation. Donor hearts were preserved for 4 hr in cold saline (4 degree C) before transplantation. Left ventricular developed pressure (LVP), basal coronary perfusion, and coronary reactivity to endothelium-dependent dilation (bradykinin, 0.1 microM) or endothelium-dependent dilation (sodium nitroprusside, 0.5 microM) were studied in isolated, buffer-perfused heart, using a modified Langendorff model. Cold preservation alone significantly reduced LVP and coronary perfusion. Coronary reactivity to bradykinin and sodium nitroprusside was also significantly impaired. In U74389G-treated donor hearts, 4 hr of cold ischemia did not alter contractile function, coronary perfusion or endothelial reactivity, although the response to sodium nitroprusside did not fully recover. In untreated recipients, in vivo reperfusion (transplantation) resulted in reduced LVP and perfusion deficits. Treating donors and recipients with U74389G improved left ventricular contractibility and coronary perfusion, although endothelium-dependent and -independent coronary reactivity remained affected. These results indicate that the lazaroid U74389G exerts significant cardioprotection during both preservation and transplantation of the heart. Donor and recipient pretreatment is mandatory for maximal efficacy with U74389G.

Improved 4- and 6-Hour Myocardial Preservation by Hypoxic Preconditioning

A brief hypoxic episode can precondition myocardium against a subsequent ischemic-reperfusion injury. The present study sought to determine whether intracellular ionic alterations, induced expression of heat-shock proteins (hsps), and/or catalase are involved in the cellular mechanisms by which hypoxic preconditioning can preserve postischemic function in a model of prolonged hypothermic storage. Two groups of isolated working rat hearts were studied: control (CON) and hypoxically preconditioned (HP) hearts. Hearts were arrested at 4 degrees C with St Thomas' cardioplegic solution and immersion-stored for either a 4- or 6-hour period. Myocardial function (ie, heart rate, aortic flow, coronary flow, developed pressure, and its first derivative dP/dtmax) was determined at baseline, after preconditioning, and during reperfusion. At similar time points, myocardial [Na+]i, [K+]i, [Mg2+]i, and [Ca2+]i were measured using an atomic absorption spectrophotometer, and the induction of hsp 70 and catalase mRNAs was assayed using Northern blot analysis. After 4 and 6 hours of hypothermic storage, aortic flow, dP/dtmax, and [K+]i were increased, whereas [Na+]i and [Ca2+]i were decreased significantly in the HP group compared with the CON group. Steady state mRNA levels of catalase and hsp 70 were increased from baseline levels only in the HP group, with a peak (2.8- and 2.4-fold versus baseline) after 4 hours of storage. Our results indicate that intracellular ionic alterations and upregulation of catalase and hsp 70 gene expression may contribute to the mechanisms underlying hypoxic preconditioning, leading to improved postischemic function during prolonged hypothermic storage of hearts.

Adenosine pretreatment for prolonged cardiac storage: an evaluation with St. Thomas' Hospital and University of Wisconsin solutions

Adenosine pretreatment has been shown to be beneficial in several models of ischemia-reperfusion. We wished to evaluate whether adenosine pretreatment is cardioprotective for prolonged cardiac storage and whether the presence of adenosine in the storage media affects the results. Isolated rodent hearts were obtained from Sprague-Dawley rats, mounted on a Langendorff apparatus, instrumented with an intraventricular balloon, and ventricularly paced at 300 beats/min. Four groups of hearts were studied in a 2 × 2 factorial experiment ( n = 8 to 12 per group). Hearts were subjected to normal perfusion or to solution supplemented with adenosine 50 μmol/L for 10 minutes followed by adenosine-free perfusion for 10 minutes. Hearts then were stored for 8 hours at 0º C in either University of Wisconsin solution (adenosine 5 mmol/L) or St. Thomas' Hospital II solution (adenosine free). Adenosine pretreatment increased tissue levels of adenosine triphosphate before storage ( p = 0.04). Nonfunction was less common after storage (1/19 versus 6/20 hearts, p < 0.05), and diastolic function was better preserved in the adenosine groups in the reperfusion phase ( p = 0.01). The beneficial effects of adenosine pretreatment were independent of which storage solution was used. Developed pressure was increased ( p < 0.05) and release of creatine kinase and lactate dehydrogenase was reduced ( p < 0.0001) in hearts treated with University of Wisconsin solution compared with those treated with St. Thomas' Hospital solution. These studies suggest that adenosine pretreatment improves recovery after prolonged hypothermic storage and that the presence of adenosine in the preservation solution does not alter the results. The experiments provide further evidence that extended myocardial protection is better enhanced with University of Wisconsin solution than with St. Thomas' Hospital II solution. (J T HORAC C ARDIOVASC S URG 1995;110:293-301)

Cardiac storage with University of Wisconsin solution and a nucleoside-transport blocker

Findings from previous investigations conducted at this institution and others have suggested that University of Wisconsin solution (UWS) is preferable for the prolonged hypothermic storage of hearts before transplantation. The benefit seen with UWS may in part be related to the inclusion of adenosine (5 mmol/L) in the UWS. To investigate whether further manipulations of adenosine metabolism might enhance myocardial protection, studies were initially conducted using cultured myocytes, followed by confirmatory experiments using isolated rat hearts. Cultured human ventricular myocytes (7 to 8 dishes/group) were stored for 12 hours at 0 degrees C in unmodified UWS or UWS supplemented with increasing concentrations (1 to 100 mumol/L) of the nucleoside-transport blocker p-nitrobenzylthioinosine. The adenosine triphosphate concentrations were found to be enhanced with nucleoside-transport inhibition, with the best results achieved with the 1- and 3-mumol/L groups (control, 3.37 +/- 0.41 nmol/micrograms DNA; UWS, 2.89 +/- 1.31 nmol/micrograms DNA; 1 mumol/L, 5.91 +/- 3.23 nmol/micrograms DNA; 3 mumol/L, 7.86 +/- 3.45 nmol/micrograms DNA; p < 0.05 versus control or UWS group). Isolated rodent hearts from Sprague-Dawley rats were prepared on a Langendorff apparatus with an intraventricular balloon and subsequently stored for 8 hours at 0 degrees C in unmodified UWS (13 hearts/group) or UWS supplemented with 1 or 3 mumol/L of p-nitrobenzylthioinosine (9 to 10 hearts/group).(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term preservation of the rat isolated heart with staurosporine and 2,3-butanedione monoxime.

The present study was designed to investigate the effectiveness of staurosporine and 2,3-butanedione monoxime (BDM) in preserving cardiac function of long-term hypothermic-stored hearts. Rat isolated hearts were perfused very slowly at 4 degrees C for 16 hr with a storage buffer solution containing staurosporine and BDM. Heart functions were then examined during 2 hr of normothermic reperfusion. Isovolumetric left ventricular-developed pressure (LVDP), its differential, heart rate, and coronary flow were measured in 5 groups of hearts: controls (fresh unstored hearts), stored drug-free hearts, stored staurosporine-treated hearts, stored BDM-treated hearts, and stored BDM + staurosporine-treated hearts. Hearts that had been perfused with staurosporine or BDM during hypothermic storage attained LVDP values that were 37% or 70%, respectively, of that shown by the control group. Hearts perfused without any drug in the storage buffer attained an LVDP value that was 20% of the control value. Heart rates of stored and then normothermically reperfused hearts were lower than, but not significantly different from, values in the control group. Coronary flow values in all stored hearts were significantly lower than the control values. Thus, BDM, and to a lesser extent staurosporine, applied during prolonged hypothermic storage improved cardiac function during normothermic reperfusion.

Cardiac storage with UW solution and glucose

Previous investigations from our institution using an isolated human cardiomyocyte model concluded that glucose supplementation of University of Wisconsin solution (UWS) was beneficial with respect to adenine nucleotide and protein recovery. We wished to confirm these results using an isolated heart model. Rodent hearts were frozen in liquid nitrogen (control) or flushed and stored in UWS for 8 hours at 0 °C or UWS supplemented with 10, 20, or 30 mmol/L glucose. Experimental hearts were assessed at end-storage or after 45 minutes of reperfusion on a Langendorff apparatus. Adenine nucleotides were assessed by high performance liquid chromatography. In parallel experiments, ventricular function was assessed before and after storage in Langendorff-perfused hearts instrumented with a left ventricular balloon. Glucose supplementation was associated with greater poststorage (20 and 30 mmol/L glucose) and postreperfusion (10, 20, and 30 mmol/L glucose) adenosine triphosphate levels than unmodified UWS. Developed pressure (expressed as a percentage of control values) was increased with 10 mmol/L glucose (75.2% ± 7.9%, mean ± standard deviation) compared with unmodified UWS (64.6% ± 6.6%; p < 0.05). Coronary flow was greater with 10 (72.6% ± 10.7%) or 20 mmol/L (71.2% ± 12.5%) versus 0 mmol/L glucose (58.6% ± 12.1%, p < 0.05). The data support previous in vitro findings and suggest that the addition of 10 mmol/L glucose to UWS is associated with enhanced recovery after prolonged hypothermic storage.

Prolonged neonatal myocardial preservation with a highly buffered low-calcium solution

Optimal methods of myocardial preservation remain controversial in the neonate. This study compared prolonged hypothermic storage of neonatal hearts with modified University of Wisconsin solution (group I) with a solution formulated to promote anaerobic glycolysis by providing proton buffering with histidine (100 mmol/L) and exogenous glucose and insulin (group II). Hearts from piglets aged 3 to 5 days were given a single dose of either solution ( n = 6 each), subjected to 20 hours of global ischemia at 4° C, and reperfused with an erythrocyte-enhanced perfusate in an isovolumic Langendorff preparation. After 1 hour of reperfusion, in comparison with hearts preserved with University of Wisconsin solution, those in group II demonstrated (mean ± standard error of the mean) greater left ventricular developed pressure (101 ± 7 versus 62 ± 9 mm Hg, p < 0.01) and the first derivative of left ventricular pressure (816 ± 23 versus 614 ± 69 mm Hg · sec -1, p < 0.05). Diastolic indices were reduced to a similar degree in the two groups. Myocardial oxygen consumption was significantly greater (38.8 ± 2.4 versus 11.8 ± 2.4 μl oxygen · min · g -1, p < 0.01) in group II hearts. Group I hearts vasoconstricted (6% increase in coronary vascular resistance) in response to an intracoronary infusion of acetylcholine (20 nmol·min -1); in contrast, acetylcholine produced coronary dilation in group II hearts (5% decrease in coronary resistance, p < 0.02) that was similar to that observed in nonischemic control hearts. These results demonstrate enhanced preservation of myocardial contractility, oxidative metabolism, and vascular function in neonatal hearts provided by a solution designed to buffer protons and promote anaerobic glycolysis during ischemia. (J THORAC CARDIOVASC SURG 1994;108:772-9)

Cardioplegic effect of University of Wisconsin solution on hypothermic ischemia of rat myocardium assessed by mitochondrial oxidative phosphorylation

The effectiveness of the University of Wisconsin solution and the Collins' M solution for preservation of rat hearts was compared by examining histologic appearance, tissue water content, and mitochondrial respiratory functions after prolonged hypothermic storage and subsequent heterotopic transplantation. Survival of transplanted hearts after 5 days of reperfusion was markedly lowered by storage in Collins' M solution for 15 hours. Hearts stored in University of Wisconsin solution for 10 hours showed no increase in myocardial necrosis after 5 days of reperfusion, whereas hearts stored in University of Wisconsin solution for 15 hours and Collins' M solution for 10 and 15 hours showed a significant increase in tissue necrosis. University of Wisconsin solution reduced tissue swelling during hypothermic storage, whereas Collins' M solution did not cause such reduction. The yield of mitochondrial protein after reperfusion was significantly decreased by storage in either solution, especially after 15 hours in Collins' M solution. Mitochondrial oxidative phosphorylation was significantly inhibited by storage, especially by storage in Collins' M solution and subsequent reperfusion. These results indicate that myocardial injury, after prolonged ischemia and reperfusion, results in a decrease in functionally and structurally intact mitochondria that is dependent on preservation conditions. University of Wisconsin solution protects isolated hearts against ischemia and reperfusion injury possibly by preventing cellular and mitochondrial deterioration.

Comparison of two experimental models for assessment of cardiac preservation

Previous studies from this institution using human cell cultures have suggested that University of Wisconsin solution is preferred for prolonged hypothermic storage for cardiac transplantation. The primary objective of this study was to evaluate the effectiveness of extended cardiac preservation with University of Wisconsin solution by assessing the time-related changes of purine metabolites using two different models of cold storage. Isolated rat hearts (n = 6/group) or human ventricular myocyte cultures (n = 7 dishes/group) were assessed after 0, 6, 12, and 24 hours in University of Wisconsin solution at 0 °C using high-performance liquid chromatography. Adenosine triphosphate content decreased from 18.1 ± 5.4 to 9.6 ± 2.7 μmol/g dried weight by 12 hours and to 1.0 ± 0.6 μmol/g by 24 hours ( p < 0.0001 by analysis of variance) in the rat model. Adenosine triphosphate content decreased from 0.64 ± 0.42 to 0.14 ± 0.11 nmol/μg DNA at 6 hours and to 0.04 ± 0.03 nmol/μg DNA by 24 hours ( p < 0.00001) in the cardiomyocytes. Inosine monophosphate content increased from 0.1 ± 0.2 to 10.8 ± 1.0 by 24 hours ( p < 0.0001) in the rat studies. Inosine monophosphate values tended to increase up to 12 hours ( p = 0.06) in the cell cultures and then declined. Adenosine concentration increased from 0.3 ± 0.3 to 2.3 ± 0.9 μmol/g at 6 hours and declined thereafter ( p < 0.0005) in the rodent hearts. Adenosine concentration increased from 0.03 ± 0.02 to 1.53 ± 0.72 nmol/μg DNA at 6 hours ( p < 0.0001) in the cardiomyocytes. Human cell cultures may represent a more sensitive model to evaluate adenine nucleotides after cardiac storage conditions and avoid species-specific changes in purine degradation. Nucleoside contents are likely increased unphysiologically with this experimental technique.

Prolonged hypothermic cardiac storage for transplantation

Cardiac storage for transplantation is currently limited to 6 hours. To better understand the metabolic changes that occur during hypothermic (4 degrees C) storage, we monitored the morphologic and metabolic changes in the canine myocardium at 0, 12, and 24 hours of storage in University of Wisconsin solution. Attempts to isolate cardiac mitochondria resulted in a progressive decline in the yield (milligrams of mitochondria per gram of heart tissue), which decreased (p less than 0.05) from 9.2 +/- 0.4 at 0 hours (control) to 4.0 +/- 0.3 after 12 hours and further decreased (p less than 0.05) to 1.9 +/- 0.2 after 24 hours of cold storage. Mitochondrial state 3 respiration fell to 64% of control after 12 hours and 28% of control after 24 hours of cold storage (p less than 0.05). Citrate synthetase activity, but not cytochrome C oxidase activity, was significantly depressed after 12 and 24 hours of cold storage. Adenosine triphosphate content decreased to 67% of control after 12 hours and 50% of control after 24 hours. After 12 hours of storage, sufficient adenosine diphosphate and monophosphate were present to permit some restoration of adenosine triphosphate, provided mitochondrial function was normal after transplantation. However, restoration of mitochondrial function and adenosine triphosphate levels sufficient to support myocardial contractility was unlikely after 24 hours of storage. This study suggests that a return of adequate cardiac function after transplantation may be possible after 12 hours of cold storage in University of Wisconsin solution but not after 24 hours of cold storage.

Effect of sodium aspartate on the recovery of the rat heart from long-term hypothermic storage

We have investigated the reported ability of aspartate to enhance greatly the cardioprotective properties of the St. Thomas' Hospital cardioplegic solution after prolonged hypothermic storage. Rat hearts (n = 8 per group) were excised and subjected to immediate arrest with St. Thomas' Hospital cardioplegic solution (2 minutes at 4 degrees C) with or without addition of monosodium aspartate (20 mmol/L). The hearts were then immersed in the same solution for 8 hours (4 degrees C) before heterotopic transplantation into the abdomen of homozygous rats and reperfusion in vivo for 24 hours. The hearts were then excised and perfused in the Langendorff mode (20 minutes). Addition of aspartate to St. Thomas' Hospital cardioplegic solution gave a small but significant improvement in left ventricular developed pressure, which recovered to 82 +/- 3 mm Hg compared with 70 +/- 2 mm Hg in control hearts (p less than 0.05). However, coronary flow and high-energy phosphate content were similar in both groups. In subsequent experiments hearts (n = 8 per group) were excised, arrested (2 minutes at 4 degrees C) with St. Thomas' Hospital cardioplegic solution containing a 0, 5, 10, 20, 30, 40, or 50 mmol/L concentration of aspartate, stored for 8 hours at 4 degrees C, and then reperfused for 35 minutes. A bell-shaped dose-response curve was obtained, with maximum recovery in the 20 mmol/L aspartate group (cardiac output, 48 +/- 5 ml/min versus 32 +/- 5 ml/min in the aspartate-free control group; p less than 0.05). However, additional experiments showed that a comparable improvement could be achieved simply by increasing the sodium concentration of St. Thomas' Hospital cardioplegic solution by 20 mmol/L. Similarly, if sodium aspartate (20 mmol/L) was added and the sodium content of the St. Thomas' Hospital cardioplegic solution reduced by 20 mmol/L, no significant protection was observed when recovery was compared with that of unmodified St. Thomas' Hospital cardioplegic solution alone. In still further studies, hearts (n = 8 per group) were perfused in the working mode at either high (greater than 80 ml/min) or low (less than 50 ml/min) left atrial filling rates. Under these conditions, if functional recovery was expressed as a percentage of preischemic function, artifactually high recoveries could be obtained in the low-filling-rate group. In conclusion, assessment of the protective properties of organic additives to cardioplegic solutions requires careful consideration of (1) the consequences of coincident changes in ionic composition and (2) the characteristics of the model used for assessment.