Recovery Of Aortic Flow Research Articles

The Effect of Increasing Donor Age on Myocardial Ischemic Tolerance in a Rodent Model of Donation After Circulatory Death.

Background.Hearts from older donors or procured via donation after circulatory death (DCD) can alleviate transplant waitlist; however, these hearts are particularly vulnerable to injury caused by warm ischemic times (WITs) inherent to DCD. This study investigates how the combination of increasing donor age and pharmacologic supplementation affects the ischemic tolerance and functional recovery of DCD hearts and how age impacts cardiac mitochondrial respiratory capacity and oxidative phosphorylation.Methods.Wistar rats (12-, 18-, and 24-mo-old) were subjected to DCD with 20-min fixed WIT. Hearts were procured, instrumented onto a Langendorff perfusion circuit, flushed with Celsior preservation solution with or without supplementation (glyceryl trinitrate [GTN]/erythropoietin [EPO]/zoniporide [Z]) and perfused (Krebs-Henseleit buffer, 37°C Langendorff 30-min, working 30-min). Cardiac functional recovery of aortic flow (AF), coronary flow (CF), cardiac output (CO), and lactate dehydrogenase release were measured. Native heart tissue (3-, 12-, and 24-mo) were assessed for mitochondrial respiratory capacity.Results.Unsupplemented 18- and 24-month DCD hearts showed a 6-fold decrease in AF recovery relative to unsupplemented 12-month DCD hearts. GTN/EPO/Z supplementation significantly increased AF and CO recovery of 18-month DCD hearts to levels comparable to supplemented 12-month hearts; however, GTN/EPO/Z did not improve 24-month DCD heart recovery. Compared to 12-month heart tissue, 24-month hearts exhibited significantly impaired mitochondrial oxygen flux at complex I, II, and uncoupled maximal respiration stage.Conclusions.Reduced ischemic tolerance after DCD was associated with increasing age. Pharmacologic supplementation improves functional recovery of rat DCD hearts but only up to age 18 months, possibly attributed to a decline in mitochondrial respiratory capacity with increasing age.

Hi1a, an ASIC1a Inhibitor, Significantly Improves the Tolerance of Donor Allografts to Ischaemia in a Rodent Model of DCD Heart Transplantation

Purpose ASIC1a has been demonstrated to play a critical role in acidosis-induced neuronal injury following cerebral ischaemia, however its role in the heart is poorly understood. Given similar pathophysiology, this project sought to investigate the cardioprotective effects of Hi1a, a novel ASIC1a inhibitor, derived from funnel web spider venom, in the context of donor allograft preservation. Methods Studies utilized a working rodent model of DCD heart preservation. Following asphyxia, hearts were retrieved from male Wistar rats (350-425g) after a 10 min warm ischaemic time (WIT) followed by 2 min stand-off, mimicking a clinical DCD withdrawal. Hearts were then flushed with either Celsior alone, Celsior + Hi1a (CHi1a), or Celsior + Hi1a, GTN and EPO (n=7/group). Allografts were reperfused ex-vivo in resting Langendorff mode for 30 min, prior to assessment of cardiac function in working mode for 30 min. Results Recovery of aortic flow (AF), pulse pressure (PP), and cardiac output (CO) was significantly improved in the CHi1a group and CHi1a + GTN, EPO group when compared to Celsior alone (p Conclusion Supplementation of Celsior with either Hi1a as a single supplement, or in combination with GTN and EPO significantly improved cardiac allograft function following warm ischaemia. Both the functional and biochemical results demonstrate profound cardioprotective effects of Hi1a. These results suggest the potential of Hi1a, or other ASIC1a inhibitors to increase the tolerable WIT during DCD retrieval, leading to further increases in heart transplant volume.

Ischaemic Tolerance and the Effect of Ageing Using a Rodent Donation after Circulatory Death (DCD) Model.

To investigate the effect of ageing on donor heart recovery after exposure to DCD withdrawal in a rodent model. Method Wistar rats (3, 12, 18 and 24 months) were anaesthetised and instrumented for invasive blood pressure and continuous heart rate/saturation monitoring using pulse oximetry. Circulatory death was induced by asphyxiation via tracheal ligation followed by injection of 500IU heparin via the renal vein. 3 month rats were exposed to 12 minutes warm ischaemia time (WIT). All other groups were exposed to 20 minutes WIT. At the end of the fixed WIT, hearts were flushed with 100mLs of celsior (C) or supplemented celsior(Cs) solution (containing erythropoietin, glyceryl trinitrate and zoniporide), followed by reperfusion on ex-vivo perfusion rig with oxygenated Krebs buffer at 37oc for 15 minutes followed by ‘working’ mode for 30mins. Aortic flow (AF), heart rate (HR) and pressure (MAP) were measured using PowerLab™; coronary flow (CF) was measured at 5 minute intervals. Results HR and MAP recovery were similar in all groups. CF and AF recovery are presented below: All results were corrected to heart weight[SVMHS1].CF was poorest in 3C group when compared to all other groups. AF recovery was superior in both 12mo groups when compared to other age groups (P<0.05); and all Cs groups show better AF recovery when compared to C only group. (P<0.05)Conclusion These results suggest that myocardial tolerance to WIT increases through adolescence into adulthood then declines through the process of ageing. It is important to note, however, all age groups showed improved recovery after WIT with pharmacological supplementation.

Improved heart function from older donors using pharmacologic conditioning strategies

Hearts from older donors are increasingly being referred for transplantation. However, these hearts are more susceptible to ischemia-reperfusion injury (IRI), reflected in higher rates of primary graft dysfunction. We assessed a strategy of pharmacologic conditioning, supplementing Celsior (Genzyme, Naarden, The Netherlands) preservation solution with glyceryl trinitrate (GTN; Hospira Australia Pty, Ltd, Mulgrave, VIC, Australia), erythropoietin (EPO; Eprex; Janssen-Cilag, North Ryde, NSW, Australia), and zoniporide (ZON; Pfizer, Inc., Groton, CT), to protect older hearts against IRI and improve graft function. Wistar rats, aged 3, 12, and 18 months old, were used to represent adolescent, 30-year-old, and 45-year-old human donors, respectively. Animals were subjected to brain death (BD) and hearts stored for 6 hours at 2° to 3°C in Celsior or Celsior supplemented with GTN+EPO+ZON. Cardiac function and lactate dehydrogenase before and after storage were assessed during ex vivo perfusion. Western blots and histopathology were also analyzed. After BD, 18-month hearts demonstrated impaired aortic flow, coronary flow, and cardiac output compared with 3-month hearts (p < 0.001 to p < 0.0001). After storage in Celsior, the recovery of aortic flow, coronary flow, and cardiac output in 18-month BD hearts was further impaired (p < 0.01 vs 3-month hearts). Percentage functional recovery of 18-month BD hearts stored in Celsior supplemented with GTN+EPO+ZON was equivalent to that of 3-month hearts and significantly improved compared with 18-month hearts stored in Celsior alone (p < 0.01 to p < 0.001), with reduced lactate dehydrogenase release (p < 0.01) and myocardial edema (p < 0.05) and elevated phosphorylated extracellular signal-related kinase 1/2 (p < 0.05) and phosphorylated Akt (p < 0.01). Older hearts are more susceptible to IRI induced by BD and prolonged hypothermic storage. Supplemented Celsior activates cell survival signaling in older hearts, reduces IRI, and enhances donor heart preservation.

Testosterone Administration Induces Protection Against Global Myocardial Ischemia

We tested the hypothesis that chronic testosterone treatment would promote a cardioprotective phenotype against ischemia/reperfusion (I/R) injury. For this study, 3-month-old F344 male rats underwent sham-surgery, orchiectomy (ORX), or ORX plus 21 days testosterone treatment (1.0 mg testosterone/day). At sacrifice, cardiac performance was assessed in a working heart model of I/R (25 min of global ischemia and 45 min of reperfusion). ORX reduced serum testosterone by approximately 98% and testosterone administration elevated serum testosterone to a concentration of 4.6-fold over that of Sham-operated controls (p<0.05). ORX did not significantly impair recovery of cardiac performance following I/R, but did increase cardiac release of lactate dehydrogenase (LDH) during pre- and post-ischemia (p<0.05). Testosterone administration prevented the ORX-induced increase in LDH during both pre- and post-ischemia and increased post-ischemic recovery of aortic flow, cardiac output, cardiac work, left ventricular developed pressure, and contractility (p<0.05) during reperfusion. Testosterone administration also increased left ventricular expression of catalase, but did not affect the expression of manganese superoxide dismutase, glutathione peroxidase, or sarcolemmal K (ATP) channel protein Kir6.2. Neither circulating nor cardiac concentrations of estradiol were altered by either treatment. We conclude that administration of high-dose testosterone confers cardioprotection through yet to be identified androgen-dependent mechanism(s).

Testosterone administration induces protection against global myocardial ischemia

We tested the hypothesis that testosterone (T) treatment affords protection against ischemia/reperfusion (I/R) injury. Orchiectomized (ORX) F344 male rats received 1.0 mg T/day for 21 days or exercise training (EX) for 10 days. Treatment groups were: Sham‐operated, ORX, ORX+T and ORX+EX. At sacrifice, cardiac performance was assessed in a working heart model of I/R (25 minutes of global ischemia and 45 minutes of reperfusion). T increased recovery of aortic flow by 2.2‐fold (p &lt; 0.05) and cardiac work by 2.0‐fold (p &lt; 0.05). ORX reduced recovery of left ventricular developed pressure (LVDP) by 42% (p &lt; 0.05). T increased recovery of LVDP by 2.8‐fold and cardiac contractility (+dP/dt) by 45% (p &lt; 0.05). ORX caused a 2.6‐fold increase in pre‐ischemic cardiac release of lactate dehydrogenase (LDH), an effect that was prevented by T (p &lt; 0.05), but not EX. T reduced post‐ischemic release of LDH release by 45% (p &lt; 0.05). EX caused a 13% increase in cardiac expression of MnSOD and T caused a 28% increase in catalase expression. Glutathione peroxidase (GPX) was not affected by either intervention. ORX reduced serum T by ~98% and T administration elevated T 4.6‐fold over Sham (p &lt; 0.05). Serum estrogen was undetectable in all groups. We conclude that that loss of natural T predisposes the heart to increased contractile dysfunction during I/R, and that high‐dose T confers cardioprotection through yet to be identified mechanism(s).

Adrenocorticotrope Hormone Fragment (4-10) Attenuates the Ischemia/Reperfusion-Induced Cardiac Injury in Isolated Rat Hearts

The aim of our study was to investigate the contribution of the adrenocorticotropic hormone fragment, ACTH (4-10), on the recovery of postischemic cardiac function. Effects of ACTH (4-10) on caspase-3 activity, cardiomyocyte and endothelial apoptosis, and HO-1 protein expression were studied. Rats were treated with various doses of ACTH (4-10), and then 12 h later, anesthetized, hearts were isolated, perfused, and subjected to 30-min ischemia followed by 120-min reperfusion. Cardiac function including heart rate, coronary flow, aortic flow, and left ventricular developed pressure were recorded. After 120-min reperfusion, 200 mug/kg of ACTH (4-10) significantly improved the recovery of aortic flow, coronary flow, and left ventricular developed pressure from their untreated control values of 15.3 +/- 0.9 ml/min, 6.5 +/- 0.9 ml/min, and 10 +/- 0.6 kPa to 20.7 +/- 1.3 ml/min, 24.8 +/- 1.8 ml/min and 13.7 +/- 0.7 kPa, respectively. Heart rate did not show significant changes during reperfusion. ACTH (4-10) treatment resulted in a reduction in infarct size, caspase 3 activity, apoptosis, and an increase in HO-1 expression. When ACTH (4-10) was given at the moment of reperfusion, the drug failed to improve the postischemic recovery of the myocardium. Thus, ACTH (4-10) can be a useful tool for the prevention of the development of ischemia/reperfusion-induced injury.

Inhibition of ischemia/reperfusion-induced damage by dexamethasone in isolated working rat hearts: the role of cytochrome c release

We investigated the contribution of dexamethasone treatment on the recovery of postischemic cardiac function and the development of reperfusion-induced arrhythmias in ischemic/reperfused isolated rat hearts. Rats were treated with 2 mg/kg of intraperitoneal injection of dexamethasone, and 24 hours later, hearts were isolated according to the ‘working’ mode, perfused, and subjected to 30 min global ischemia followed by 120 min reperfusion. Cardiac function including heart rate, coronary flow, aortic flow, and left ventricular developed pressure were recorded. After 60 min and 120 min reperfusion, 2 mg/kg of dexamethasone significantly improved the postischemic recovery of aortic flow and left ventricular developed pressure from their control values of 10.7 ± 0.3 ml/min and 10.5 ± 0.3 kPa to 22.2 ± 0.3 ml/min (p < 0.05) and 14.3 ± 0.5 kPa (p < 0.05), 19.3 ± 0.3 ml/min (p < 0.05) and 12.3 ± 0.5 kPa (p < 0.05), respectively. Heart rate and coronary flow did not show a significant change in postischemic recovery after 60 or 120 min reperfusion. In rats treated with 0.5 mg/kg of actinomycin D injected i.v., one hour before the dexamethasone injection, suppressed the dexamethasone-induced cardiac protection. Electrocardiograms were monitored to determine the incidence of reperfusion-induced ventricular fibrillation. Dexamethasone pretreatment significantly reduces the occurrence of ventricular fibrillation. Cytochrome c release was also observed in the cytoplasm. The results suggest that the inhibition of cytochrome c release is involved in the dexamethasone-induced cardiac protection.

Myocardial protection by ischemic preconditioning and δ-opioid receptor activation in the isolated working rat heart

Objective: δ-Opioid receptors are involved in the cardioprotective effect of ischemic preconditioning. This study was designed (1) to assess the protective capacities of ischemic preconditioning and the synthetic δ-opioid receptor agonist D-Ala2-D-Leu5 enkephalin (DADLE) in a functionally oriented experimental model of ischemia and reperfusion and (2) to assess whether the effects of both protective measures are similarly blocked by naloxone, a nonspecific δ-opioid receptor antagonist. Methods: Sixty-four isolated working rat hearts were subjected to 45 minutes of hypothermic ischemia at 30°C followed by 25 minutes of normothermic reperfusion. Rats were pretreated with DADLE (1 mg/kg body weight intravenously), naloxone (3 mg/kg body weight intravenously), or a combination thereof within 60 minutes before onset of isolated heart perfusion. During the preischemic perfusion period, 8 hearts per group were preconditioned by one cycle of 5 minutes of normothermic global ischemia and subsequent reperfusion whereas another 8 served as nonpreconditioned controls. The postischemic functional recovery of hearts and their creatine kinase leakage were determined. Results: Pretreatment with DADLE and ischemic preconditioning improved the postischemic recovery of aortic flow when compared with nonpreconditioning (57.7% ± 4.0% and 60.8% ± 4.3% vs 40.0% ± 4.2% of preischemic baseline value, P ||.001). Combined pretreatment with DADLE before ischemic preconditioning afforded additional aortic flow recovery compared with pretreatment with DADLE alone (68.6% ± 3.3% vs 57.7% ± 4.0% of preischemic baseline value; P =.038). With combined pretreatment, early postischemic creatine kinase release was lower than control in hearts without pretreatment (0.48 ± 0.11 vs 0.80 ± 0.12 IU/5 minutes per heart; P =.001). Naloxone abolished the beneficial functional effects of pretreatment with DADLE and ischemic preconditioning. Conclusions: Pharmacologic activation of δ-opioid receptors affords improvement of functional protection in isolated working rat hearts similar to that conferred by classic ischemic preconditioning. The combination of both pretreatments reduces ischemic cellular damage and further adds to postischemic functional recovery. These changes are reversed by naloxone, an observation providing evidence that ischemic preconditioning involves signaling through opioid receptors.J Thorac Cardiovasc Surg 2001;122:986-92

The push-and-pull mechanism to scavenge redox-active transition metals: A novel concept in myocardial protection

Objective: Traces of redox-active transition metals such as iron and copper play an important role in free radical formation during postischemic reperfusion of the heart. Two studies were conducted to assess the efficacy of the complexes of desferrioxamine with zinc or gallium to prevent this aspect of reperfusion injury. Methods: In study I, isolated working rat hearts (n = 96) were subjected to 2 hours of hypothermic arrest at 10°C induced by use of St Thomas' Hospital cardioplegic solution II supplemented with desferrioxamine, zinc-histidinate, zinc-desferrioxamine, gallium-nitrate, or gallium-desferrioxamine. In study II, isolated nonworking rat hearts (n = 23) were subjected to normothermic regional (10 minutes) or global (35 minutes) unprotected ischemia. In this study, the perfusate was supplemented with gallium-desferrioxamine during preischemic and postischemic periods. Results: In study I, the addition of desferrioxamine, zinc-histidinate, or gallium-nitrate to St Thomas' Hospital solution II improved postischemic aortic flow recovery. When the binary complexes zinc-desferrioxamine or gallium-desferrioxamine were added, however, functional recovery was further enhanced significantly. In study II, high-performance liquid chromatography analyses of tissue from postischemic hearts exposed to unsupplemented perfusate revealed a marked increase of malondialdehydes. In hearts perfused with perfusate supplemented with gallium-desferrioxamine, however, tissue malondialdehyde concentrations were significantly smaller, indicating reduced free radical formation. Conclusions: The data suggest synergistic protection by the complexes of the iron chelator desferrioxamine with zinc or gallium. The single components neutralize transition metals by 2 different but complementary push-and-pull mechanisms, thereby leading to an inhibition of metal-mediated site-specific free radical formation and improvement of postischemic cardiac function. (J Thorac Cardiovasc Surg 2001;121:1169-78)

Risk of low calcium and high magnesium in continuous warm hyperkalemic cardioplegia

Background. The recent introduction of operations on a warm heart has prompted clinical reports on the usefulness of continuous blood cardioplegia, but no in-depth basic evaluation of continuous cardioplegia has been done. The cardioprotective effects of magnesium (Mg) and calcium (Ca) in continuous warm hyperkalemic crystalloid cardioplegic solutions were investigated in an isolated rat heart model. Methods. Isolated rat hearts were arrested for 180 minutes at 37°C with a continuous warm hyperkalemic (20 mmol/L) modified Krebs-Henseleit bicarbonate buffer solution containing 1.2, 8.0, or 16.0 mmol/L of Mg and 0.1 to 2.5 mmol/L of Ca in different concentrations. Recovery of cardiac function and tissue damage were estimated. Results. For each Mg concentration, the percentage recovery of aortic flow generated dose-response curves depending on Ca concentration. However, as Mg concentration increased, the recovery of aortic flow decreased in the groups with 0.5 mmol/L of Ca or less. Conclusions. In continuous warm cardioplegia the combination of low Ca and high Mg concentration caused severe cardiac injury, and normal Ca concentration avoids cardiac injury regardless of Mg concentrations.

The advantages of normocalcemic continuous warm cardioplegia over low calcemic cardioplegia in myocardial protection.

The effects of changing the calcium content of a continuous warm hyperkalemic crystalloid cardioplegia (CWCP) were investigated in an isolated rat heart preparation. The hearts were divided into eight groups of six each. A control group consisted of fresh nonarrested hearts and the remaining seven groups consisted of hearts perfused with continuous hyperkalemic (20 mM) modified Krebs-Henseleit bicarbonate buffer solution with calcium concentrations of 0.1, 0.3, 0.5, 1.0, 1.5, 2.0, or 2.5 mM, for either 180 or 240 min at 37 degrees C. In the hearts arrested for 180 min, there were no significant differences in postarrest cardiac functions between the control group and any of the groups perfused with calcium concentrations of 0.5 mM or more. With a calcium concentration of 0.1 mM, the calcium paradox was provoked. The change in the calcium content of CWCP perfused for 240 min significantly affected myocardial protection. Maximum aortic flow recovery, of 74.7% +/- 2.7%, and minimum CK release, of 15.7 +/- 2.4 IU/15 min/g dry weight, were observed in hearts perfused with a calcium concentration of 1.5 mM. The calcium paradox occurred even at a calcium concentration of 0.3 mM; therefore, normal calcium concentrations should be maintained in cardiac surgery to prevent cardiac injury.

Long-term myocardial preservation: effects of hyperkalemia, sodium channel, and NA +/K +/2CL - cotransport inhibition on extracellular potassium accumulation during hypothermic storage

Objectives: We previously demonstrated improved myocardial preservation with polarized (tetrodotoxin-induced), compared with depolarized (hyperkalemia-induced), arrest and hypothermic storage. This study was undertaken to determine whether polarized arrest reduced ionic imbalance during ischemic storage and whether this was influenced by Na +/K +/2Cl - cotransport inhibition. Methods: We used the isolated crystalloid perfused working rat heart preparation (1) to measure extracellular K + accumulation (using a K +-sensitive intramyocardial electrode) during ischemic (control), depolarized (K + 16 mmol/L), and polarized (tetrodotoxin, 22 μmol/L) arrest and hypothermic (7.5°C) storage (5 hours), (2) to determine dose-dependent (0.1, 1.0, 10 and 100 μmol/L) effects of the Na +/K +/2Cl - cotransport inhibitor, furosemide, on extracellular K + accumulation during polarized arrest and 7.5°C storage, and (3) to correlate extracellular K + accumulation to postischemic recovery of cardiac function. Results: Characteristic triphasic profiles of extracellular K + accumulation were observed in control and depolarized arrested hearts; a significantly attenuated profile with polarized arrested hearts demonstrated reduced extracellular K + accumulation, correlating with higher postischemic function (recovery of aortic flow was 54% ±4% [ P = .01] compared with 39% ±3% and 32% ±3% in depolarized and control hearts, respectively). Furosemide (0.1, 1.0, 10, and 100 μmol/L) modified extracellular K + accumulation by –18%, –38%, –0.2%, and +9%, respectively, after 30 minutes and by –4%, –27%, +31%, and +42%, respectively, after 5 hours of polarized storage. Recovery of aortic flow was 53% ±4% (polarized arrest alone), 56% ±8%, 70% ±2% ( P = .04 vs control), 69% ±4% ( P = .04 vs control), and 65% ±3% ( P = .04 vs control), respectively. Conclusions: Polarized arrest was associated with a reduced ionic imbalance (demonstrated by reduced extracellular K + accumulation) and improved recovery of cardiac function. Further attenuation of extracellular K + accumulation (by furosemide) resulted in additional recovery. (J Thorac Cardiovasc Surg 1999;118:123-34)

Effects of amiodarone on cardiac function and mitochondrial oxidative phosphorylation during ischemia and reperfusion.

This study was carried out in order to determine if the efficiency of amiodarone, a class III antiarrhythmic agent, is associated with changes in mitochondrial oxidative phosphorylation. A population of 30 rats were treated with amiodarone (100 mg/kg/day) for 5 days. A second population receiving only vehicle was used as control. The hearts were perfused according to the working mode. After 15 min of normoxic perfusion, the left main coronary artery was ligated and the ligation was maintained for 20 min. The ligation was removed and reperfusion continued for a further 30 min. The electrocardiogram was monitored continuously. At the end of perfusion, the ischemic and non ischemic areas were visually separated and mitochondria were harvested from each area. Their oxidative and energy metabolism were assessed with palmitoylcarnitine as substrate in 2 respiration media differing in their free calcium concentration (0 or 0.34 microm). In normoxic conditions, amiodarone treatment increased the cardiac metabolic efficiency (mechanical work to oxygen consumption ratio). The local ischemia decreased the aortic and coronary flows without modifying the cardiac metabolic efficiency. Amiodarone treatment maintained the aortic flow at a significantly higher value; the duration of severe arrhythmias was significantly decreased by the drug. The reperfusion of the ischemic area allowed the partial recovery of fluid dynamics. The coronary flow was restored to 89% of the pre ischemic value. Conversely, the aortic flow never exceeded that measured at the end of ischemia, partly due to the important development of severe arrhythmias. The recovery of aortic flow and metabolic efficiency during reperfusion was improved by amiodarone treatment; ventricular tachycardia and fibrillation duration were reduced. In the mitochondria issued from the normoxic area, the energy metabolism was not altered by the amiodarone treatment, but the presence of calcium in the respiration medium modified the oxidative phosphorylation. The divalent cation slightly decreased the state III respiration rate and increased noticeably the state IV respiration rate. This was associated with an important mitochondrial AMP production and maintenance of ADP in the respiration medium. This energy wasting was reported to decrease the mitochondrial metabolic efficiency. After an ischemia-reperfusion sequence, mitochondrial oxidation phosphorylation was reduced and amiodarone treatment amplified this decrease. This was presumably due to an increased mitochondrial calcium accumulation. Thus, the beneficial properties of amiodarone during reperfusion are supposed to be due to a protection against the deleterious effect of excess matrix calcium on mitochondrial energy metabolism.

Individual effects of dietary EPA and DHA on the functioning of the isolated working rat heart

The aim of this study was to evaluate the effects of dietary pure eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on the physiology of the heart in normoxic conditions and during postischemic reperfusion. These effects were compared with those of dietary n-6 polyunsaturated fatty acids (PUFA). Rats were fed a diet containing either sunflower seed oil (75 g·kg-1, SSO group), or a mixture of EPA (20:5 n-3) ethyl ester and SSO (10:90, EPA group), or a mixture of DHA (22:6 n-3) ethyl ester and SSO (10:90, DHA group), or a mixture of EPA + DHA ethyl esters and SSO (4.2:5.8:90, e+D group) for 6 weeks. The hearts were then perfused according to the working mode. The perfusion was maintained either in normoxic conditions or stopped for 17 min (global zero-flow ischemia) and restored for 33 min (reperfusion). The aortic and coronary flows, aortic developed pressure, and electrocardiogram were continuously monitored. When rats were fed a diet containing either EPA and (or) DHA, the n-6/n-3 PUFA ratio of cardiac phospholipids decreased. The proportion of arachidonic acid was reduced more with DHA than dietary EPA. In the EPA group, the percentage of DHA was lower than in the DHA group, but the percentage of EPA and docosapentaenoic acid (22:5 n-3) was higher. These changes in membrane fatty acid composition altered the cardiac function. In normoxic conditions, the coronary flow was higher in the SSO group than in the DHA and EPA groups. The heart rate was lower in the DHA and e+D groups than in the EPA and SSO groups. The aortic flow, cardiac output, and aortic developed pressure were not affected. During postischemic reperfusion, the recovery of aortic flow, coronary flow, and aortic developed pressure was similar in the four groups. A slightly improved recovery of cardiac function was noticed in the EPA group, but the difference was not significant. Feeding rats 5% fish oil + 5% SSO instead of 10% SSO for 8 weeks increased the incorporation of EPA in cardiac phospholipids and favored the recovery (+120%) of aortic flow during postischemic reperfusion. In conclusion, the beneficial effect of dietary fish oil on the recovery of cardiac pump activity during reperfusion was not observed with DHA or EPA alone. It appears to be positively related to the accumulation of EPA in membrane phospholipids. The dietary conditions favouring EPA accumulation remain to be determined.Key words: dietary polyunsaturated fatty acids, myocardial ischemia, reperfusion.

Decreasing sarcoplasmic reticular calcium gives rise to myocardial protection--the effect of thapsigargin for myocardial protection under conditions of normothermia

Deceasing sarcoplasmic reticular (SR) calcium may contribute to the myocardiac protection against ischemia and reperfusion-induced injury. Therefore, using the isolated working rat heart model, we investigated the effect of Thapsigargin (TH)-induced SR calcium diminution on the myocardial protection when added either before onset of ischemia or at time of reperfusion under conditions of normothermic ischemia. Hearts (n = 6/group) from male Wistar rats were aerobically (37 degrees C) perfused (20 min) with bicarbonate buffer. In the experimental protocol A, this was followed by a 3 min infusion of St. Thomas' Hospital cardioplegic solution No. 2 (STS) containing various concentrations of TH. Hearts were then subjected to 34 min of normothermic (37 degrees C) global ischemia and 35 min of reperfusion (15 min Langendorff, 20 min working). Reperfusion cardiac functions at 20 min of working perfusion was measured and compared with the preischemia values. STS added to 0.1 and 0.25 mumol/L TH improved recovery of aortic flow after 20 min reperfusion from 47 +/- 3% in the TH free controls to 62 +/- 3, 63 +/- 2% (n = 6) (p < 0.05). There was no difference in creatine kinase (CK) leakage during Langendorff reperfusion between the TH treated groups and the control group. In the experimental protocol B, 3 min of cardioplegia without TH and 34 min of ischemia (37 degrees C) were followed by a 10 min Langendorff reperfusion with various concentrations of TH, then 10 min Langendroff reperfusion for washing out, and 20 min working reperfusion. When TH was added to reperfusate the recovery of aortic flow did not change, 0.5 mumol/L TH group had the detelious effect. Thus, TH, when added to the cardioplegia, enhanced myocardial protection. We conclude that lessened uptake of Ca2+ into sarcoplasmic reticulum by inhibitors of the Ca(2+)-ATPase pump can decrease ischemia and reperfusion-induced injury.

Superior Recovery of Hypertrophied Rat Myocardium After Cardioplegic Arrest

Background. Although cardioplegic protection of the hypertrophied heart remains a clinical challenge, we have previously observed enhanced recovery in rat hearts with pressure-overload hypertrophy induced by aortic banding. We investigated whether this unexpected result is found in other models of hypertrophy. Methods. Hearts with hypertrophy induced by aortic banding or administration of desoxycorticosterone acetate were each compared with age-matched sham-operated and nonoperated controls. Spontaneously hypertensive rats and Wistar-Kyoto controls were also compared. We evaluated left ventricular isomyosin distribution by gel electrophoresis and recovery of isolated working rat hearts arrested at 8°C for 2 hours. Results. The percentage of V 3 isomyosin in hearts with hypertrophy from aortic banding or administration of desoxycorticosterone acetate was increased compared with the control groups. Recovery of aortic flow in all three groups of hypertrophied hearts was at least as good or better than their respective controls. There were no significant differences in ATP or glycogen between hypertrophied and control hearts before or after arrest. Conclusions. Enhanced recovery of hypertrophied hearts is not specific to a single model. This level of recovery may be supported by induction of a “fetal genetic program,” exemplified in the rat by the shift in isomyosin from predominantly V 1 to the more efficient V 3 isoform, which occurs in pressure-overloaded hearts.

Comparison of Polarized and Depolarized Arrest in the Isolated Rat Heart for Long-term Preservation

Hypothermic hyperkalemic cardioplegic solutions are currently used for donor heart preservation. Hyperkalemia-induced depolarization of the resting membrane potential (Em) may predispose the heart to Na+ and Ca2+ loading via voltage-dependent "window currents," thereby exacerbating injury and limiting the safe storage duration. Alternatively, maintaining the resting Em with a polarizing solution may reduce ionic movements and improve postischemic recovery; we investigated this concept with the reversible sodium channel blocker tetrodotoxin (TTX) to determine (1) whether polarized arrest was more efficacious than depolarized arrest during hypothermic long-term myocardial preservation and (2) whether TTX induces and maintains polarized arrest. The isolated crystalloid-perfused working rat heart preparation was used in this study. Preliminary studies determined an optimal TTX concentration of 22 micromol/L and an optimal storage temperature of 7.5 degrees C. To compare depolarized and polarized arrest, hearts were arrested with either Krebs-Henseleit (KH) buffer (control), KH buffer containing 16 mmol/L K+, or KH buffer containing 22 micromol/L TTX and then stored at 7.5 degrees C for 5 hours. Postischemic recovery of aortic flow was 13+/-4%, 38+/-2%, and 48+/-3%* (*P<.05 versus control and 16 mmol/L K+), respectively. When conventional 3 mol/L KCl-filled intracellular microelectrodes were used, Em gradually depolarized during control unprotected ischemia to approximately -55 mV before reperfusion, whereas arrest with 16 mmol/L K+ caused rapid depolarization to approximately -50 mV, where it remained throughout the 5-hour storage period. In contrast, in 22 micromol/L TTX-arrested hearts, Em remained more polarized, at approximately -70 mV, for the entire ischemic period. Blockade of cardiac sodium channels by TTX during ischemia maintained polarized arrest, which was more protective than depolarized arrest, possibly because of reduced ionic imbalance.

Myocardial Protection of Contractile Function After Global Ischemia by Physiologic Estrogen Replacement in the Ovariectomized Rat

The role of physiologic estrogen levels (pg) on post-ischemic myocardial function was studied in the isolated working rat heart without (n=28, experiment No. 1) or with (n=15, experiment No. 2) preconditioning. For experiment No. 1, female ovariectomized rats were treated with placebo (n=19) or 17β-estradiol (E2,n=9; chronic E2), and 14 days later hearts were removed and perfused with modified Krebs–Henseleit bufferin vitro. In nine placebo-treated rats, E2 was administered at 20 min prior to ischemia (acute E2). The hearts were subjected to 15 min of global ischemia and 20 min of reflow. In experiment No. 2, ovariectomized rats were treated with placebo (n=8) or 17β-E2 (chronic E2,n=7). In this experiment, hearts were first preconditioned and then subjected to 20 min of sustained ischemia followed by 20 min of reflow. Global ischemia was produced by clamping the aorta and restricting left atrial flow so that coronary flow was reduced to zero; hemodynamics were continuously monitored throughout the study. Aortic flow, coronary flow, cardiac output, and dP/dtwere assessed at baseline, at the end of the ischemic period, and during reflow. The severity of ischemia was measured by post-ischemic release of lactate, lactate dehydrogenase, and creatine kinase and was similar among all groups in each study. In experiment No. 1, recovery of aortic flow, cardiac output, and dP/dtfollowing reperfusion, was significantly improved in rats treated with chronic E2 (P<0.05); acute E2 had no significant benefit. Post-ischemic recovery of coronary flow was not significantly affected. In experiment No. 2, chronic E2 treatment also significantly improved post-ischemic recovery of cardiac function in preconditioned hearts when compared with controls (P<0.05). In summary, E2 replacement in ovariectomized rats improves contractile function following global ischemia and reflow; cardioprotection by estrogen was observed over and above that conferred by ischemic preconditioning. Since the cardioprotective effect of E2 was independent of a significant improvement in coronary flow a direct effect of the hormone on the cardiac myocytes is postulated.

Relative susceptibility of endothelium and myocardial cells to ischaemia-reperfusion injury.

We have investigated the timescale, extent and sequence of endothelial, myocardial and smooth muscle cell dysfunction following ischaemia-reperfusion in the isolated rat heart. Myocardial function in the form of aortic flow was evaluated in the working heart preparation. Vasodilatation induced by endothelium-dependent agent 5-hydroxytryptamine (5-HT) and endothelium-independent substance glyceryl trinitrate (GTN) was used to assess endothelial and smooth muscle function, respectively. The percentage recovery of 5-HT response and aortic flow (endothelial vs. myocardial function) plus 5-HT response and GTN effect (endothelial function vs. smooth muscle response) after a period of ischaemia was statistically analysed by Wilcoxon's signed rank test. A possible correlation between structural and functional changes in coronary vascular endothelium after ischaemia was also explored by comparing endothelial dysfunction with morphological assessment of endothelial damage seen after ischaemia. The experiments were performed at two clinically relevant temperatures of 20 degrees C and 4 degrees C. There was no significant difference in the percentage recovery of aortic flow and 5-HT response or in 5-HT response and GTN effect after 30 min of unprotected ischaemia at 4 degrees C. The same duration of ischaemia at 20 degrees C led to significantly better recovery of GTN effect as compared with 5-HT response, but there was no difference in the recovery of aortic flow and 5-HT response. Sixty minutes of unprotected ischaemia at both temperatures caused significantly better recovery of aortic flow and GTN effect as compared with 5-HT response; 60 min of ischaemia protected by an infusion of a cardioplegic solution (protected ischaemia) at 4 degrees C had no significant effect on the recovery of the three parameters. Periods of 90, 120, 180 and 240 min of protected ischaemia at 4 degrees C, and 60, 90 and 120 min of protected ischaemia at 20 degrees C resulted in significantly better recovery of aortic flow compared with 5-HT response and of GTN effect as compared to 5-HT response. This demonstrates varying susceptibility of different cell types to ischaemic injury and highlights the strong vulnerability of endothelium to ischaemic damage as compared with myocytes and smooth muscle cells. Furthermore, there was lack of correlation between post-ischaemic endothelial dysfunction and microscopically assessed structural damage.