Hypothermic Heart Preservation Research Articles

(44) - 12-Hour Hypothermic Heart Preservation in a Pig Model

(44) - 12-Hour Hypothermic Heart Preservation in a Pig Model

CARC17: Outcomes in Heart Transplant Recipients with Sex Mismatched Organs: A Subgroup Analysis of the GUARDIAN-Heart Registry

Background: Donor and recipient sex mismatch in heart transplantation is considered a risk factor for poor outcomes. The mechanisms for this are poorly understood. We sought to assess whether a male to female or female to male donor mismatch influences outcomes following transplants using donor hearts preserved with the SherpaPak Cardiac Transport System (CTS). The CTS provides controlled hypothermic heart preservation during transportation which may abate cold injury to the donor heart. Methods: The GUARDIAN-Heart registry is an international, multicenter registry assessing outcomes following heart transplantation using donor organs preserved with CTS compared to standard ice transportation (ICE). A subgroup analysis of sex-mismatched donors and recipients resulted in cohorts of 121 ICE transplants compared to 141 CTS transplants. Summary statistics were used for comparing the groups. Further analysis with ongoing enrollment will be available for presentation. Results: The baselines of the two groups were similar, including the proportion of female to male mismatches (57% (ICE) vs 56%, (CTS), p = 0.87) and the percent undersized by >15% (17.4% (ICE) vs 22.5% (CTS), p = 0.33), except there was significantly longer total ischemic time in transplants using CTS by 26 minutes (195.2 vs 221.2 minutes, p = 0.004) and distance traveled to procure the organs was 44% farther (251.6 vs 445.7 miles, p < 0.001, ICE vs CTS, respectively). Post-transplant, the incidence of severe PGD was similar (11.7% vs 7.8%, p = 0.29, ICE vs CTS, respectively), as was the total length of ICU and hospital stay. The CTS cohort demonstrated a significant improvement in both 30 day (95% vs 100%, p = 0.01) and in-hospital survival (92.6% vs 99.3%, p = 0.005, ICE vs CTS, respectively). Conclusions: Despite significantly longer ischemic times, use of the SherpaPak CTS resulted in improved early survival when compared to heart transplants using standard ice transport.

Myocardial Substrate Oxidation and Tricarboxylic Acid Cycle Intermediates During Hypothermic Machine Perfusion

BackgroundThe optimal substrate for hypothermic machine perfusion preservation of donor hearts is unknown. Fatty acids, acetate, and ketones are preferred substrates of the heart during normothermic perfusion, but cannot replete the tricarboxylic acid (TCA) cycle directly. Propionate, an anaplerotic substrate, can replenish TCA cycle intermediates and may affect cardiac metabolism. The purpose of this study was to determine myocardial substrate preferences during hypothermic machine perfusion and to assess if an anaplerotic substrate was required to maintain the TCA cycle intermediate pool in perfused hearts. MethodsGroups of rat hearts were perfused with carbon-13 (13C)-labeled substrates (acetate, β-hydroxybutyrate, octanoate, with and without propionate) at low and high concentrations. TCA cycle intermediate concentrations, substrate selection, and TCA cycle flux were determined by gas chromatography/mass spectroscopy and 13C magnetic resonance spectroscopy. ResultsAcetate and octanoate were preferentially oxidized, whereas β-hydroxybutyrate was a minor substrate. TCA cycle intermediate concentrations except fumarate were higher in substrate-containing perfusion groups compared with either the no-substrate perfusion group or the no-ischemia control group. ConclusionsThe presence of an exogenous, oxidizable substrate is required to support metabolism in the cold perfused heart. An anaplerotic substrate is not essential to maintain the TCA cycle intermediate pool and support oxidative metabolism under these conditions.

Hypothermic preservation of rat hearts using antifreeze glycoprotein.

Antifreeze proteins are an effective additive for low-temperature preservation of solid organs. Here, we compared static hypothermic preservation with and without antifreeze glycoprotein (AFGP), followed by nonfreezing cryopreservation of rat hearts. The heart was surgically extracted and immersed in one of the cardioplegia solutions after cardiac arrest. Control rat hearts (n=6) were immersed in University of Wisconsin (UW) solution whereas AFGP-treated hearts (AFGP group) (n=6) were immersed in UW solution containing 500 ?g/ml AFGP. After static hypothermic preservation, a Langendorff apparatus was used to reperfuse the coronary arteries with oxygenated Krebs-Henseleit solution. After 30, 60, 90, and 120 min, the heart rate (HR), coronary flow (CF), cardiac contractile force (max dP/dt), and cardiac diastolic force (min dP/dt) were measured. Tissue water content (TWC) and tissue adenosine triphosphate (ATP) levels in the reperfused preserved hearts were also assessed. All the parameters were compared between the control and AFGP groups. Compared with the control group, the AFGP group had significantly (p<0.05) higher values of the following parameters: HR at 60, 90, and 120 min; CF at all four time points; max dP/dt at 90 min; min dP/dt at 90 and 120 min; and tissue ATP levels at 120 min. TWC did not differ significantly between the groups. The higher HR, CF, max dP/dt, min dP/dt, and tissue ATP levels in the AFGP compared with those in control hearts suggested that AFGP conferred superior hemodynamic and metabolic functions. Thus, AFGP might be a useful additive for the static/nonfreezing hypothermic preservation of hearts.

Cold-inducible RNA binding protein agonist enhances the cardioprotective effect of UW solution during extended heart preservation.

In heart transplantation, time restriction is an unavoidable thorny problem during cardiac transport. Cold storage is an important organ preservation method in donor heart transport. Cold-inducible RNA binding protein (CIRBP) has been proven to play a protective role under cold stress. In this study, we investigated the role of CIRBP in hypothermic cardioprotection during heart preservation in UW solution and explored a new approach to extend the heart preservation time. Cirbp-knockout (Cirbp-/- ), Cirbp-transgenic (Cirbp-Tg), and wild-type rats were, respectively, randomized into two groups based on various heart preservation times (6 or 12-hour group) (n=8 per group). After preservation in UW solution, all hearts were mounted on aLangendorff apparatus andunderwent measurement of cardiac parameters, histological analysis, and molecular study. Within the 6-hour preservation group, no significant difference was found in cardiac functions and histological changes between different rat species. However, after 12 hours of preservation, Cirbp-/- rat hearts showed more apoptosis and worse cardiac function, but less apoptosis and better cardiac function were observed in Cirbp-Tg rat hearts. Furthermore, we found CIRBP-mediated cardiac ubiquinone (CoQ10 ) biosynthesis plays an important role in extending heart preservation, and ubiquinone biosynthesis protein COQ9 was an essential down-stream regulator during this process. Finally, we found that zr17-2, a CIRBP agonist, could enhance the expression of CIRBP, which further enhances the synthesis of CoQ10 and promotes scavenging of reactive oxygen species and ATP production to extend heart preservation. This study demonstrated that CIRBP-enhanced CoQ10 biosynthesis during hypothermic heart preservation and zr17-2-supplemented UW solution could be a promising approach to ameliorate heart damage and extend heart preservation during cardiac transport.

Utilization of New Heart Preservation Technology in the Era of New Organ Allocation and Extended Ischemic Times

Current ice bag heart preservation technique is associated with wide fluctuations of organ temperature, resulting in sub-optimal preservation and freeze injury. These shortcomings may be particularly impactful with increased ischemic times given increasing retrieval distances associated with new organ allocation. In comparison, the SherpaPakTM Cardiac Transport System is designed for the static hypothermic preservation of donor hearts within a controlled temperature range. This method avoids excess cooling and optimizes preservation during transportation. The purpose of this study is to assess the outcomes of heart transplantation using the static hypothermic preservation (SHP) system as compared with conventional ice bag preservation (IP). Retrospective cohort analysis was performed on adult patients undergoing heart transplantation between 6/30/2018 and 09/02/2019 at a single institution. Donor and recipient baseline characteristics and post-transplant outcomes were compared between patients receiving organs with SHP versus IP for donor heart transport. Patients who underwent multiple organ transplantation were excluded. Of 48 consecutive patients undergoing heart transplantation, 20 (41.7%) organs were transported using SHP and 28 (58.3%) using IP. SHP was used for older recipients (61.5 vs 57.5 years, p=0.02), and longer donor heart cold ischemic times (4.35 vs 3.49 hours, Figure1, p=0.018). There were no 30-day mortalities. Despite longer ischemic times, there was no difference in the incidence of any primary graft dysfunction (PGD) (14.3% vs 15.0%, p=1) or severe PGD requiring extracorporeal membrane oxygenation (10.7% vs 5.0%, Table1, p=0.63) between SHP and IP. There was no worsening in clinical outcomes with SHP despite longer ischemic times and older recipient ages. With new heart allocation changes, SHP may be beneficial in cases with further transport distances, by mitigating the effect of prolonged ischemic times.

Functional recovery after dantrolene-supplementation of cold stored hearts using an ex vivo isolated working rat heart model.

The ryanodine receptor antagonist dantrolene inhibits calcium release from the sarcoplasmic reticulum and reduces cardiac ischaemia-reperfusion injury (IRI) in global warm ischaemia models however the cardioprotective potential of dantrolene under hypothermic conditions is unknown. This study addresses whether the addition of dantrolene during cardioplegia and hypothermic storage of the donor heart can improve functional recovery and reduce IRI. Using an ex vivo isolated working heart model, Wistar rat (3 month and 12 month) hearts were perfused to acquire baseline haemodynamic measurements of aortic flow, coronary flow, cardiac output, pulse pressure and heart rate. Hearts were arrested and stored in Celsior preservation solution supplemented with 0.2–40 μM dantrolene for 6 hours at 4°C, then reperfused (15 min Langendorff, 30 min working mode). In 3-month hearts, supplementation with 1 μM dantrolene significantly improved aortic flow and cardiac output compared to unsupplemented controls however lactate dehydrogenase (LDH) release and contraction bands were comparable. In contrast, 40 μM dantrolene-supplementation yielded poor cardiac recovery, increased post-reperfusion LDH but reduced contraction bands. All 3-month hearts stored in dantrolene displayed significantly reduced cleaved-caspase 3 intensities compared to controls. Analysis of cardioprotective signalling pathways showed no changes in AMPKα however dantrolene increased STAT3 and ERK1/2 signaling in a manner unrelated to functional recovery and AKT activity was reduced in 1 μM dantrolene-stored hearts. In contrast to 3-month hearts, no significant improvements were observed in the functional recovery of 12-month hearts following prolonged storage in 1 μM dantrolene. Conclusions: Dantrolene supplementation at 1 μM during hypothermic heart preservation improved functional recovery of young, but not older (12 month) hearts. Although the molecular mechanisms responsible for dantrolene-mediated cardioprotection are unclear, our studies show no correlation between improved functional recovery and SAFE and RISK pathway activation.

Heat shock protein 90 mediates anti-apoptotic effect of diazoxide by preventing the cleavage of Bid in hypothermic preservation rat hearts

Successful organ preservation is the premise for clinical organ transplantation. The present study investigated whether heat shock protein 90 (Hsp90) is important in the anti-apoptotic effect of diazoxide in hypothermic preservation rat hearts. Isolated rat hearts were preserved in Celsior solution, with or without diazoxide, for 3 to 9 hours, followed by 60 minutes of reperfusion. Cell apoptosis was assessed by terminal deoxynucleotide transferase-mediated deoxy uridine triphosphate nick-end labeling. The left ventricular developed pressure (LVDP) was recorded. Expression of Hsp90 protein and cleavage of Bid were detected by Western blot and polymerase chain reaction. After hypothermic preservation for 3 to 9 hours, the LVDP recovery rate significantly decreased and cardiomyocyte apoptosis index increased in a time-dependent manner. When compared with the 9-hour preservation group, Celsior solution supplemented with diazoxide significantly enhanced the LVDP recovery rate and decreased the apoptosis index. The cleavage of Bid increased after 9 hours of hypothermic preservation, which was inhibited by Celsior solution supplemented with diazoxide. Hypothermic preservation of rat hearts for 9 hours decreased the expression of Hsp90, whereas diazoxide supplementation significantly increased the expression of Hsp90. The Hsp90 inhibitor 17-allylamino-17-demethoxy-geldanamycin inhibited the diazoxide-induced decrease in cleavage of Bid, improvement of cardiac function, and decrease of apoptosis. Hsp90 inhibitor had no effect on the diazoxide-induced increase of total Cx43 protein expression in hearts preserved 9 hours, but inhibited the diazoxide-induced increase of mitochondrial Cx43 protein level. Hsp90 might mediate diazoxide-induced cardioprotection against apoptosis in hypothermic preservation heart by preventing the cleavage of Bid.

Extending myocardial viability during heart preservation with cyclosporine A.

Hypothermic preservation (PRES) of donor hearts is limited to 12-14 hours for complete functional recovery after reperfusion. In a canine heterotopic heart transplant model, 50% to 60% functional recovery returned after 18 hours of PRES with University of Wisconsin (UW) solution. Concomitant with functional changes were marked increases in apoptotic cells at 2 (2.69%) and 6 (5.98%) hours of reperfusion with a concomitant decrease in lamin B1 (2% and 7.6%, respectively) with no evidence of necrotic cells. These results suggested that blockade of apoptosis may prolong myocardial viability during PRES and reperfusion. Donor hearts were subjected to 18 and 24 hours of PRES (2 degrees C to 4 degrees C) with and without cyclosporine A (CyS) treatment (apoptosis blocker). CyS was given to the donor animal (10 mg/kg), in the PRES solution (10(-5) mol/L), slowly infused during the PRES period (1 mL/min), and also to the recipient animal (2.5 mg/kg). After 18 hours of PRES with CyS, function returned to 100% within 1 hour and stayed at this level throughout a 6-hour recovery period. Apoptotic myocytes were reduced (55%) after 18 hours PRES with CyS treatment, and 6-hour reperfusion lamin B1 was reduced to only 3.7%. Twenty-four hour PRES in UW resulted in no functional recovery. However, after CyS treatment, functional recovery returned to 100% after 4 hours of reperfusion. Adenosine triphosphate (ATP) and creatine phosphate (CP) concentrations were surprisingly the same with or without CyS treatment at 18 hours and lower with 24 hours. Use of CyS in the PRES solution prolongs myocardial viability during donor heart PRES. The mechanism of action may be associated with the mitochondrial permeability transition (MPT) pore via cyclophilin D binding.

Diazoxide supplemented Celsior solution improves hypothermic heart preservation effect in rat through activation of mitochondrial ATP-sensitive potassium channel

Successful heart preservation is critical for clinical heart transplantation, but even the most successful heart preservation method can only preserve the heart in a time range of approximately 4 h. In the present study, diazoxide (DE)-containing cardioplegia solution was used as the hypothermic heart preservation solution, and hearts were preserved for different time periods before reperfusion. Five groups were randomly divided into the control group, DE groups with different concentrations of DE (15, 30, and 45 μM), and the 5-HD + DE group containing 30 μM DE and 100 μM of 5-hydroxydecanoate (5-HD). Each group was further divided according to the storage time: 3 h and 8 h subgroups. Hemodynamic parameters were monitored continually and myocardial injury markers lactate dehydrogenase (LDH) and creatine kinase (CK) were measured before arrest and during reperfusion. After reperfusion, myocardium water content, myocardium apoptosis, and myocardial mitochondrial superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were measured. All data were analyzed by independent samples t-test. Our results showed that diazoxide treatment improved impaired hemodynamics parameters and decreased the leakage of myocardial enzymes during reperfusion, compared to the control group. Moreover, diazoxide treatment significantly alleviated myocardial edema, decreased the number of TUNEL-positive cardiomyocytes, prevented the loss of mitochondrial SOD activity, and depressed the mitochondrial MDA content. This study indicated that mitoKATP channel opener diazoxide could significantly enhance myocardial protection during long-term hypothermic preservation, and the inhibition of oxidative stress-induced myocardium apoptosis seems to be the mechanism involved in the protective action on the mitoKATP channel.

Diazoxide supplemented Celsior solution improves hypothermic heart preservation effect in rat through activation of mitochondrial ATP-sensitive potassium channel

Diazoxide supplemented Celsior solution improves hypothermic heart preservation effect in rat through activation of mitochondrial ATP-sensitive potassium channel

Controlled reperfusion after hypothermic heart preservation inhibits mitochondrial permeability transition-pore opening and enhances functional recovery

We investigated whether low-pressure reperfusion may attenuate postischemic contractile dysfunction, limits necrosis and apoptosis after a prolonged hypothermic ischemia, and inhibits mitochondrial permeability transition-pore (MPTP) opening. Isolated rats hearts (n = 72) were exposed to 8 h of cold ischemia and assigned to the following groups: 1) reperfusion with low pressure (LP = 70 cmH(2)O) and 2) reperfusion with normal pressure (NP = 100 cmH(2)O). Cardiac function was assessed during reperfusion using the Langendorff model. Mitochondria were isolated, and the Ca(2+) resistance capacity (CRC) of the MPTP was determined. Malondialdehyde (MDA) production, caspase-3 activity, and cytochrome c were also assessed. We found that functional recovery was significantly improved in LP hearts with rate-pressure product averaging 30,380 +/- 1,757 vs. 18,000 +/- 1,599 mmHg/min in NP hearts (P < 0.01). Necrosis, measured by triphenyltetrazolium chloride staining and creatine kinase leakage, was significantly reduced in LP hearts (P < 0.01). The CRC was increased in LP heart mitochondria (P < 0.01). Caspase-3 activity, cytochrome c release, and MDA production were reduced in LP hearts (P < 0.001 and P < 0.01). This study demonstrated that low-pressure reperfusion after hypothermic heart ischemia improves postischemic contractile dysfunction and attenuates necrosis and apoptosis. This protection could be related to an inhibition of mitochondrial permeability transition.

Taurine prevents myocardial ischemia/reperfusion-induced oxidative stress and apoptosis in prolonged hypothermic rat heart preservation

Taurine (2-aminoethanesulfonic acid) is a potent antioxidant and inhibits cell apoptosis in ischemic reperfusion injury. In this study we evaluated whether addition of taurine to St. Thomas' cardioplegic solution enhances its myocardial protective effects in prolonged hypothermic heart preservation in rats. Hearts isolated from male Sprague-Dawley rats were mounted on a Langendorff apparatus to estimate baseline cardiac function, then arrested and stored in St. Thomas' cardioplegic solution, with taurine (10 mM; taurine group, n = 8) or without taurine (control group, n = 8), for 6 h at 4 degrees C. After storage, the hearts were reperfused and heart rate (HR), coronary flow (CF), left ventricular developed pressure (LVP), and positive maximum left ventricular developing pressure (max LV dp/dt) were measured. The LV tissue was examined immunohistochemically for determining DNA oxidative stress and cell apoptosis. Compared with control groups, recovery of LVP (P < 0.001), max LV dp/dt (P < 0.001), and coronary flow (P < 0.001) were significantly enhanced, whereas glutamic oxaloacetic transaminase (P < 0.01), lactate dehydrogenase (P < 0.05), creatine phosphate kinase (P < 0.01), 8-hydroxy-2'-deoxyguanosine index (P < 0.01), caspase-3 mRNA expression (P < 0.05), and percentage of TUNEL-positive cardiomyocytes (P < 0.05) were reduced in the taurine group. Addition of taurine to St. Thomas' cardioplegic solution improved cardiac function recovery for prolonged hypothermic rat heart preservation by suppressing DNA oxidative stress and cell apoptosis.

Protective effect of mitochondrial ATP-sensitive potassium channel opener on rat heart during hypothermic preservation

To investigate whether the mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) opener diazoxide as an additive to cardioplegia solution could enhance myocardial protection during hypothermic preservation of the rat heart. The Langendorff model of isolated rat heart was used. After equilibrium, the hearts were stored in Celsior cardioplegia solution at 4 degree with or without supplement of diazoxide for 3 or 8 h followed by 60 minutes reperfusion. The recovery of cardiac contractile function, myocardial enzyme leakage in the coronary effluent, and myocardial water content were determined. The myocardial ultrastructure was also observed. (1) Treatment of diazoxide improved the recovery of left ventricular developed pressure and decreased the leakage of myocardial enzymes, lactate dehydrogenase (LDH) and creatine kinase (CK), at the 2nd and 4th minute of reperfusion of rat heart after hypothermic preservation for 3 h. (2) After hypothermic preservation for 8 h, diazoxide improved the recovery of left ventricular developed pressure and decreased the leakage of myocardial enzymes (LDH, CK and glutamic oxalic transaminase) during reperfusion. Moreover, left ventricular end-diastolic pressure was significantly lower in diazoxide-treated hearts than that of hearts in Celsior solution. (3) Diazoxide significantly decreased the water content of myocardium and increased coronary flow of the hearts compared with those in control after hypothermic preservation for 8 h. (4) Impairment of myocardial ultrastructure after 8 h hypothermic preservation was alleviated in hearts treated with 30 mol/L diazoxide. (5) The cardiac effects of 30 mol/L diazoxide were attenuated by a mitoK(ATP) blocker 5-hydroxydecanoate (100 micromol/L). Diazoxide as a supplementation in cardioplegia solution could enhance myocardial protection during hypothermic heart preservation via opening of mitochondrial K(ATP) channel.

Supplementation of Nucleoside-Nucleotide Mixture Enhances Functional Recovery and Energy Metabolism Following Long-Time Hypothermic Heart Preservation

OG-VI, a well-balanced mixture of nucleoside and nucleotides, has been demonstrated to have a favorable effect on energy metabolism. In this study, we tested the hypothesis that addition of OG-VI to the University of Wisconsin solution can improve the cardiac functional recovery following long-time hypothermic preservation. Forty-two male Wistar rats were randomized into four groups. After 30-min of isolated working heart perfusion, the rat hearts were arrested with St. Thomas cardioplegic solution and preserved at 4 degrees C in saline, OG-VI, UW, and UW+OG-VI, respectively. After 12-h of preservation, the hearts were reperfused for 60-min during which the recovery of cardiac functions were monitored continuously. Myocardial adenine nucleotides were analyzed using high-performance liquid chromomatograph. In the UW+OG-VI group, the recovery of cardiac output, coronary flow, aortic flow, rate-pressure product, left ventricle stroke volume, and stroke work were significantly higher than other groups (P < 0.05). Furthermore, all phosphate high-energy compounds were significantly higher in the UW+OG-VI group than in the other groups (P < 0.05). Coronary vascular resistance and myocardial wet/dry weight ratio were obviously lower in the UW+OG-VI group, compared to the other groups (P < 0.05). Heart function was better recovered when nucleoside-nucleotide mixture was added to UW solution during long-time hypothermic rat heart preservation. The mechanism is not totally clear, but enhancement of high-energy phosphate production is possible.

Impact of 24 h continuous hypothermic perfusion on heart preservation by assessment of oxidative stress.

Despite investigating numerous solutions, additives, and techniques over the last two decades, extending donor heart preservation beyond 4-6 h has not been achieved. Hypothermic heart preservation (HP) induces oxidative stress (OS) with reactive oxygen species (ROS) production, causing DNA cleavage and impairing repair. Quantification of cardiomyocyte concentrations of DNA damage by-products (8-oxoG) and mismatch repair enzymes (MYH, OGG-1, MSH2) reflects the severity of OS. If increased repair enzyme production is insufficient to repair injury, cell death occurs and functional outcomes are impacted. We investigated continuous hypothermic perfusion (CHP), a new form of HP, and the mechanism of injury associated with hypothermic storage, by assessing functional outcome and OS after allotransplantation of canine hearts. Fourteen canine hearts were harvested using standard techniques after baseline echocardiograms and haemodynamic parameters were obtained. The hearts were implanted after 24 h CHP (n = 10) or 4 h static preservation (SP; n = 4). After weaning from cardiopulmonary bypass (CPB), recipients were kept alive for 6 h. Repeat echocardiograms and haemodynamic parameters were obtained. Quantification of MYH, OGG-1, and MSH2 concentrations were performed on biopsies using immunohistochemistry and Western blot analysis. Twelve out of 14 hearts (8/10 CHP; 4/4 SP) were successfully weaned on moderate inotropic support. Post-implant echocardiogram, completed in 6/10 CHP and 2/4 SP hearts, demonstrated hyperdynamic function and normal wall motion. The expression and activity of DNA repair enzymes was identical between normal baseline and CHP hearts. CHP reduces OS associated with prolonged hypothermic preservation and may allow longer preservation periods without compromising function. CHP offers several potential advantages: (1) resuscitation of non-beating heart donor organs, (2) time for HLA tissue typing, (3) facilitate interventions improving graft function, and (4) increased organ sharing.

Permeability of fructose-1,6-bisphosphate in liposomes and cardiac myocytes.

Fructose-1,6-bisphosphate (FBP) helps preserve heart and other organs under ischemic conditions. Previous studies indicated that it can be taken up by various cell types. Here we extended observations from our group that FBP could penetrate artificial lipid bilayers and be taken up by cardiac myocytes, comparing the uptake of FBP to that of L-glucose. Using liposomes prepared by the freeze-thaw method, FBP entered about 200-fold slower than L-glucose. For liposomes of either soybean or egg lipids, 50 mM FBP enhanced the permeability of FBP itself, with little effect on general permeability (measured by uptake of L-glucose). In experiments with isolated cardiac myocytes at 21 degrees C, FBP uptake exceeded the uptake of L-glucose by several fold and appeared to equilibrate by 60 min. There was both a saturable component at micromolar levels and a nonsaturable component which dominated at millimolar levels. The saturable component was inhibited by Pi and by other phosphorylated sugars, though with lower affinity than FBP. Both saturable and nonsaturable uptakes were also observed at 3 degrees C. The results indicate that FBP enters myocytes not by simple penetration through the lipid bilayer, but via at least two distinct protein-dependent processes. The uptake could lead to intracellular effects important in hypothermic heart preservation.

Mitochondrial permeability transition-pore inhibition enhances functional recovery after long-time hypothermic heart preservation.

Long-time preservation of the donor heart before transplantation is associated with mitochondrial damages resulting in functional deterioration after transplantation, although the exact mechanism behind this is still uncertain. Here, we have demonstrated the opening of a nonspecific pore in the inner membrane of the mitochondria, the mitochondrial permeability transition pore (PTP), as one of the reasons responsible for this functional deterioration. After 30 minutes of perfusion with the Krebs Henseleit buffer at 37 degrees C in working mode, hearts from Wistar rats were arrested with ice-cold St. Thomas cardioplegic solution and preserved University of Wisconsin solution at 4 degrees C with or without inhibiting the PTP with cyclosporin A (CsA) (0.2 microM). After 12 hours, the hearts were reperfused for 60 minutes at 37 degrees C with or without perfusing the hearts during first 15 minutes of reperfusion with PTP openers lonidamine (30 microM) or atractyloside (20 microM). Inhibiting PTP with CsA resulted in (a) significant recovery of cardiac functions, (b) well-preserved myocardial adenosine triphosphate (ATP) levels (P<0.001), (c) less myocardial water content (P<0.01), (d) less mitochondrial swelling and cytochrome C release, and (e) up-regulation of Bcl-2 expression compared with the control hearts without PTP inhibition. These effects are completely inhibited on opening the PTP before preservation, resulting in poor recovery of cardiac functions, loss of myocardial ATP, and severe mitochondrial swelling. The present study demonstrates the potential role of mitochondrial PTP after long- time hypothermic preservation of the heart, and therefore, regulation of PTP would prolong the preservation time of donor hearts.

Using fructose-1,6-diphosphate during hypothermic rabbit-heart preservation: a high-energy phosphate study

Background: In this study, we evaluated the effects of fructose-1,6-diphosphate (FDP) on high-energy phosphate metabolism during 18-hour hypothermic rabbit-heart preservation. Methods: Under general anesthesia and artificial ventilation, hearts from 42 adult New Zealand white rabbits were harvested, flushed, and preserved in St. Thomas solution at 4 oC for 18 hours. In the study group ( n = 15), FDP (5 mmol/liter) was added to the St. Thomas solution, whereas in the control group ( n = 17), fructose (5 mmol/liter) was added. Another 10 hearts did not undergo hypothermic storage, but were used as the normal group for high-energy phosphate concentration comparison. Results: After 18 hours of hypothermic preservation, myocardial high-energy phosphate content decreased in both preservation groups. In the study group, left ventricular adenosine triphosphate (ATP) content was 33% of that in the normal hearts, but in the control group, ATP decreased to 14% of normal. Adenosine diphosphate (ADP) content, energy charge, and ATP-to-ADP ratio showed similar decreases. The high-energy phosphate profile (content in the atria and ventricles and the ratio of ATP to ADP to AMP) was maintained in the study group but not in the control group. High-energy phosphate metabolites such as inosine monophosphate (IMP), inosine, and hypoxanthine increased in both preservation groups , but the increase was more prominent in the control group. Conclusion: Adding FDP to St. Thomas solution attenuated the depletion of high-energy phosphate concentration in the preserved hearts. This difference was especially prominent in the left and right ventricles. The protective effect of FDP during hypothermic heart preservation deserves further study.

Potential role of mitochondrial permeability transition pore following long-time hypothermic heart preservation

Potential role of mitochondrial permeability transition pore following long-time hypothermic heart preservation