Relative contribution of endogenous opioids to myocardial ischemic tolerance

BACKGROUND: Opioid preconditioning by exogenous opioids experimentally protects the myocardium against ischemia/reflow injury. Additionally, endogenous opioid peptides released during ischemia also enhance ischemic tolerance. Promiscuous opioid receptor agonists conceal the differential contribution of the mu, delta, and kappa opioid subtypes. This study compared the impact of selective delta and kappa opioid receptor antagonists on postischemic functional and metabolic recovery. Also measured were changing levels of peptides dynorphin B and met-enkephalin during ischemia/reflow injury. MATERIALS AND METHODS: Using the rabbit Langendorff model, the functional recovery of control hearts (following 2 h of global ischemia) was compared to hearts pretreated with delta antagonist NTB (1 microM) or kappa antagonist, nor-BNI (1 microM). Measures included percentage of return of isovolumetric developed pressure (LVDP), myocardial oxygen consumption (MVO(2)) and coronary flow (CF). In additional studies, untreated hearts were harvested at baseline, following ischemia, or following 5 or 45 min of reflow. Tissue concentrations of met-enkephalin and dynorphin B were measured by RIA. RESULTS: After 45 min of reflow, hearts pretreated with either NTB or nor-BNI showed impaired functional recovery by a decrease in LVDP (P < 0.05); however, MVO(2) or CF were unaffected. RIA data shows that baseline levels of both peptides are similar and increase significantly during ischemia, but reflow dynorphin levels drop far below baseline, while met-enkephalin returns to baseline. CONCLUSION: Antagonism of both delta and kappa opioid receptor subtypes equally contributes to impaired left ventricular function, independent of altered perfusion or metabolic rate. Endogenous kappa-receptor agonists may contribute primarily during ischemia or early reflow, since low late reflow dynorphin content did not correlate with altered functional recovery.     

Delta opioid receptors and low temperature myocardial protection

Background. Cardiac surgery continues to be limited by an inability to achieve complete myocardial protection. This may result from the use of hypothermic cardioplegia. Interestingly, the subcellular changes of animal hibernation parallel the altered biology of induced hypothermic myocardial ischemia, but are well tolerated by hibernated mammalian myocardium. Evidence indicates this protection is mediated by activation of the delta opioid receptor, which elicits profound metabolic effects at the whole animal, organ, and cell level. In this study, we sought to determine if pentazocine, with agonist activity at the delta opioid receptor, could improve myocardial recovery following global ischemia over a wide range of temperatures.

Methods. Isolated rabbit hearts received either standard cardioplegia or were pretreated with racemic, d or l isomer pentazocine. Hearts were then subjected to 2 hours at 34°C, or 3.5 hours at 20°C, or 4 hours at 10°C of cardioplegic ischemia and reperfused. Functional recovery was compared to controls.

Results. Isovolumic developed pressure, coronary flow, oxygen consumption, and ultrastructural preservation were enhanced with pentazocine delta opioid mediated protection, which appears to be additive to standard cardioplegia, even at low temperatures.

Conclusions. Teleologically, delta opioid protection parallels animal hibernation, which occurs from 34° down to 0°C. The use of delta opioid receptor agonists may have important clinical implications for cardiac surgery and deserves further study.     

Bradykinin pretreatment improves ischemia tolerance of the rabbit heart by tyrosine kinase mediated pathways

BACKGROUND: Depressed myocardial performance is an important clinical problem after open-heart surgery. We hypothesized that: (1) pretreating the heart with bradykinin improves postischemic performance, and (2) bradykinin activates protein tyrosine kinase (TK). METHODS: Twenty-seven adult rabbit hearts underwent retrograde perfusion with Krebs-Henseleit buffer (KHB) followed by 50 min of 37 degrees C cardioplegic ischemia with St. Thomas' cardioplegia solution (StTCP). Ten control hearts received no pretreatment. Ten bradykinin-pretreated hearts received a 10-minute infusion of 0.1 microM bradykinin-enriched KHB and cardioplegic arrest with 0.1 microM bradykinin-enriched StTCP. Seven others received 40 microM Genistein (Research Biochemicals, Natick, MA), a selective inhibitor of TK, added to both the 0.1-microM bradykinin-enriched KHB and 0.1-microM bradykinin-enriched StTCP solutions. RESULTS: Bradykinin pretreatment significantly improved postischemic myocardial performance and coronary flow (CF) compared with control (left ventricular developed pressure: 53 +/- 5 vs 27 +/- 4 mm Hg; +dP/dt(max): 1,025 +/- 93 vs 507 +/- 85 mm Hg/s; CF: 31 +/- 3 vs 22 +/- 2 mL/min; p < 0.05). Inhibition of TK with Genistein prevented this improvement in myocardial function, resulting in recovery equivalent to untreated controls. CONCLUSIONS: Bradykinin pretreatment may be an important new strategy for improving myocardial protection during heart surgery. The molecular mechanism of action may be similar to those activated by ischemic preconditioning.     

Protein kinase C isoform-dependent myocardial protection by ischemic preconditioning and potassium cardioplegia

OBJECTIVE: Ischemic preconditioning combined with potassium cardioplegia does not always confer additive myocardial protection. This study tested the hypothesis that the efficacy of ischemic preconditioning under potassium cardioplegia is dependent on protein kinase C isoform. METHODS: Isolated and crystalloid-perfused rat hearts underwent 5 cycles of 1 minute of ischemia and 5 minutes of reperfusion (low-grade ischemic preconditioning) or 3 cycles of 5 minutes of ischemia and 5 minutes of reperfusion (high-grade ischemic preconditioning) or time-matched continuous perfusion. These hearts received a further 5 minutes of infusion of normal buffer or oxygenated potassium cardioplegic solution. The isoform nonselective protein kinase C inhibitor chelerythrine (5 micromol/L) was administered throughout the preischemic period. All hearts underwent 35 minutes of normothermic global ischemia followed by 30 minutes of reperfusion. Isovolumic left ventricular function and creatine kinase release were measured as the end points of myocardial protection. Distribution of protein kinase C alpha, delta, and epsilon in the cytosol and the membrane fractions were analyzed by Western blotting and quantified by a densitometric assay. RESULTS: Low-grade ischemic preconditioning was almost as beneficial as potassium cardioplegia in improving functional recovery; left ventricular developed pressure 30 minutes after reperfusion was 70 +/- 15 mm Hg (P <.01) in low-grade ischemic preconditioning and 77 +/- 14 mm Hg (P <.001) in potassium cardioplegia compared with values found in unprotected control hearts (39 +/- 12 mm Hg). Creatine kinase release during reperfusion was also equally inhibited by low-grade ischemic preconditioning (18.2 +/- 10.6 IU/g dry weight, P <.05) and potassium cardioplegia (17.6 +/- 6.7 IU/g, P <.01) compared with control values. However, low-grade ischemic preconditioning in combination with potassium cardioplegia conferred no significant additional myocardial protection; left ventricular developed pressure was 80 +/- 17 mm Hg, and creatine kinase release was 14.8 +/- 11.0 IU/g. In contrast, high-grade ischemic preconditioning with potassium cardioplegia conferred better myocardial protection than potassium cardioplegia alone; left ventricular developed pressure was 121 +/- 16 mm Hg (P <.001), and creatine kinase release was 8.3 +/- 5.8 IU/g (P <.05). Chelerythrine itself had no significant effect on functional recovery and creatine kinase release in the control hearts, but it did inhibit the salutary effects not only of low-grade and high-grade ischemic preconditioning but also those of potassium cardioplegia. Low-grade ischemic preconditioning and potassium cardioplegia enhanced translocation of protein kinase C alpha to the membrane, whereas high-grade ischemic preconditioning also enhanced translocation of protein kinase C delta and epsilon. Chelerythrine inhibited translocation of all 3 protein kinase C isoforms. CONCLUSIONS: These results suggest that myocardial protection by low-grade ischemic preconditioning and potassium cardioplegia are mediated through enhanced translocation of protein kinase C alpha to the membrane. It is therefore suggested that activation of the novel protein kinase C isoforms is necessary to potentiate myocardial protection under potassium cardioplegia.     

Optimal hypothermic preservation of arrested myocardium in isolated perfused rabbit hearts: a 31P NMR study

The purpose of this study was to (1) relate myocardial high-energy phosphate stores to functional recovery after ischemia and reperfusion, (2) assess the bioenergetics and functional influence of clinically relevant myocardial hypothermia, and (3) examine tissue pH as an independent indicator of postischemic recovery of function. Rabbit hearts were perfused via a modified Langendorff technique, monitored for developed pressure (DP) and left ventricular end-diastolic pressure (LVEDP) via an isovolumic left ventricular balloon catheter, and placed in a Brucker NMR magnet (4.7 tesla) to measure phosphocreatine (PCr), adenosine triphosphate (ATP), and pH. Hearts underwent 1 hour of global ischemia at 7 degrees, 17 degrees, 27 degrees and 37 degrees C initiated by one dose of K+ cardioplegia followed by 30 minutes of reperfusion. After reperfusion, DP (expressed as a percentage of preischemic control) and LVEDP (mm Hg) in 7 degrees and 17 degrees C hearts were no different (96 + 5% vs 97 +/- 3%; 5 +/- 2 mm Hg vs 6 +/- 2 mm Hg; p = NS), but were better (p less than 0.01) than 27 degree hearts (72 +/- 6%, 17 +/- 6 mm Hg) and 37 degree hearts (31 +/- 7%, 60 +/- 6 mm Hg). PCr was severely depleted in all groups. ATP was 90 +/- 7% and 87 +/- 5% of preischemic control in the 7 degree and 17 degree hearts, which was significantly better than the 68 +/- 3% and 21 +/- 3% in the 27 degree and 37 degree groups (p less than 0.01). The pH at end ischemia was 6.83, 6.89, 6.54, and 5.86 for the 7 degree, 17 degree, 27 degree, and 37 degree hearts, respectively (7 degrees vs 27 degrees or 37 degrees, p less than 0.01; 17 degrees vs 27 degrees or 37 degrees, p less than 0.01). Linear regression of DP on end-ischemic ATP (EIATP) and end-ischemic pH revealed: DP = 0.96 (EIATP) + 20 (r = 0.92) and DP = 60 (pH) -317 (r = 0.86). We conclude that (1) end-ischemic ATP predicts recovery of ventricular function, and, furthermore, there appears a threshold ATP concentration (80% of control) below which full recovery of function will not occur; (2) end-ischemic pH predicts recovery of ventricular function; (3) 7 degrees C hypothermic ischemia does not cause a clinically significant cold injury; and (4) in a single-dose crystalloid cardioplegia model, end-ischemic pH is linearly related to recovery of function (r = 0.86).     

Myocardial protection with oxygenated esmolol cardioplegia during prolonged normothermic ischemia in the rat

OBJECTIVE: We previously showed that arrest with multidose infusions of high-dose (1 mmol/L) esmolol (an ultra-short-acting beta-blocker) in oxygenated Krebs-Henseleit buffer (esmolol cardioplegia) provided complete myocardial protection after 40 minutes of normothermic (37 degrees C) global ischemia in isolated rat hearts. In this study we investigated the importance of oxygenation for protection with esmolol cardioplegia, compared it with that of St Thomas' Hospital cardioplegia, and determined the protective efficacy of multidose esmolol cardioplegia for extended ischemic durations. METHODS: Isolated rat hearts (n = 6/group) were perfused in the Langendorff mode at constant pressure (75 mm Hg) with oxygenated Krebs-Henseleit bicarbonate buffer at 37 degrees C. The first part of the first study had four groups: (i) multidose (every 15 minutes) oxygenated (95% oxygen/5% carbon dioxide) Krebs-Henseleit buffer during 60 minutes of global ischemia, (ii) multidose deoxygenated (95% nitrogen/5% carbon dioxide) Krebs-Henseleit buffer during 60 minutes of global ischemia, (iii) multidose oxygenated esmolol cardioplegia during 60 minutes of global ischemia, and (iv) multidose deoxygenated esmolol cardioplegia during 60 minutes of global ischemia. The second part of the first study had three groups: (v) multidose St Thomas' Hospital solution during 60 minutes of global ischemia, (vi) multidose oxygenated St Thomas' Hospital solution during 60 minutes of global ischemia, and (vii) multidose oxygenated esmolol cardioplegia during 60 minutes of global ischemia. In the second study, hearts were randomly assigned to 60, 75, 90, or 120 minutes of global ischemia and at each ischemic duration were subjected to multidose oxygenated constant flow or constant pressure infusion of (i) Krebs-Henseleit buffer (constant flow), (ii) Krebs-Henseleit buffer (constant pressure), (iii) esmolol cardioplegia (constant flow), or (iv) esmolol cardioplegia (constant pressure). All hearts were reperfused for 60 minutes, and recovery of function was measured. RESULTS: Multidose infusion of oxygenated esmolol cardioplegia completely protected the hearts (97% +/- 5%) after 60 minutes of 37 degrees C global ischemia. Deoxygenated esmolol cardioplegia was significantly less protective (45% +/- 8%). Oxygenation of St Thomas' Hospital solution did not alter its protective efficacy in this study (70% +/- 4% vs 69% +/- 7%). Infusion of esmolol cardioplegia at constant pressure provided complete protection for 60, 75, and 90 minutes (104% +/- 5%, 95% +/- 5%, and 95% +/- 3%, respectively), whereas protection with constant-flow esmolol cardioplegic infusion was significantly decreased at ischemic durations longer than 60 minutes. This decrease in efficacy of constant-flow esmolol cardioplegia was associated with increasing coronary perfusion pressure leading to myocardial injury. CONCLUSIONS: Oxygenation of esmolol cardioplegia (Krebs-Henseleit buffer plus 1.0 mmol/L esmolol) was essential for optimal myocardial protection. Multidose infusion of oxygenated esmolol cardioplegia provided good myocardial protection during extended periods of normothermic ischemia. Esmolol cardioplegia may provide an efficacious alternative to hyperkalemia.     

Is protection of ischemic neonatal myocardium by cardioplegia species dependent?

Hypothermia combined with pharmacologic cardioplegia protects the globally ischemic adult heart, but this benefit may not extend to children, resulting in poor postischemic recovery of function and increased mortality. The relative susceptibilities to ischemia modified by hypothermia alone and by hypothermia plus cardioplegia were assessed in isolated perfused neonatal (3- to 4-day-old) rabbit and pig hearts. Hearts were perfused aerobically with Krebs buffer solution in the working mode for 30 minutes and aortic flow was recorded. This was followed by 3 minutes of hypothermic (14 degrees C) coronary perfusion with either Krebs or St. Thomas' Hospital cardioplegic solution No. 2 followed by hypothermic (14 degrees C) global ischemia (rabbits 2, 4, and 6 hours; pigs 2 and 4 hours). Hearts were reperfused for 15 minutes in the Langendorff mode and 30 minutes in the working mode, and recovery of postischemic aortic flow was measured. Hypothermia alone provided excellent protection of the ischemic neonatal rabbit heart, with recovery of aortic flow after 2 and 4 hours of ischemia at 91% +/- 4% and 87% +/- 5% (mean +/- standard deviation) of its preischemic value. Recovery after 6 hours of ischemia was depressed to 58% +/- 9% of its preischemic value. Ischemic neonatal pig hearts protected with hypothermia alone recovered 94% +/- 3% of preischemic aortic flow after 2 hours; none was able to generate flow after 4 hours. St. Thomas' Hospital solution No. 2 decreased postischemic aortic flow after 4 hours of ischemia in rabbit hearts from 87% +/- 5% to 70% +/- 7% (p less than 0.05, hypothermia alone versus hypothermia plus cardioplegia) but improved postischemic recovery of aortic flow in pig hearts after 4 hours of ischemia from 0 to 73% +/- 13% (p less than 0.0001, hypothermia alone versus hypothermia plus cardioplegia). This effect was dose related in both species. We conclude that the neonatal pig heart is more susceptible to ischemia modified by hypothermia alone than the neonatal rabbit and that St. Thomas' Hospital solution No. 2 improves postischemic recovery of function in the neonatal pig but decreases it in the neonatal rabbit. This species-dependent protection of the neonatal heart may be related to differences in the extent of myocardial maturity at the time of study.     

Potassium-channel opener cardioplegia is superior to St. Thomas' solution in the intact animal.

BACKGROUND: In isolated hearts, the potassium-channel opener pinacidil is an effective cardioplegic agent. This study tested the hypothesis that pinacidil is superior to St. Thomas' solution in the more clinically relevant intact animal. METHODS: Sixteen pigs were placed on full cardiopulmonary bypass. Hearts underwent 2 hours of global ischemia (10 degrees to 15 degrees C). Either St. Thomas' or 100 micromol/L pinacidil was administered every 20 minutes (10 mL/kg). Preischemic and postreperfusion slopes of the preload-recruitable stroke work relationship were determined. Changes in myocardial adenine nucleotide levels and cellular ultrastructure were analyzed. RESULTS: Pinacidil cardioplegia resulted in an insignificant change in the slope of the preload-recruitable stroke work relationship (40.6+/-2.1 mm Hg/mm before ischemia and 36.5+/-3.7 mm Hg/mm after ischemia; p = 0.466). In contrast, St. Thomas' solution resulted in a significant decrease in the slope after reperfusion (34.3+/-5.5 mm Hg/mm and 13.5+/-2.3 mm Hg/mm; p = 0.003). Adenine nucleotide levels, myocardial tissue water, and ultrastructural changes were similar between groups. CONCLUSIONS: Pinacidil ameliorated myocardial stunning associated with traditional hyperkalemic cardioplegia without causing significant differences in cellular metabolism.     

Improved myocardial preservation during global ischemia by continuous retrograde coronary sinus perfusion

To investigate whether retrograde continuous low-pressure perfusion of the coronary sinus could deliver cardioplegic solutions with oxygen and substrate beyond stenoses and result in improved myocardial preservation, we subjected 41 canine hearts to 90 minutes of ischemia with an occlusion on the circumflex coronary artery. There were four groups: Group I, antegrade (aortic root) crystalloid cardioplegia every 30 minutes during ischemia; Group II, antegrade plus topical cooling; Group III, continuous retrograde perfusion; Group IV, same as Group III, with an oxygenated perfluorocarbon. All solutions had a PO2 of 400 to 500 mm Hg. Intramyocardial oxygen and carbon dioxide tensions (PO2 and PCO2) and mean myocardial temperatures were monitored during ischemia, and left ventricular (LV) function was assessed before ischemia and after reperfusion. After global ischemia, the circumflex occlusion was released and the hearts reperfused. Following 60 minutes of reperfusion, isovolumic developed pressure returned to 36% +/- 4% and 41% +/- 5% of preischemic levels, respectively, in Groups I and II. By contrast, Groups III and IV (retrograde perfusion) had a significantly greater percent of recovery (78% +/- 5% and 73% +/- 5%). Circumflex area intramyocardial PO2 fell 20 and 25 mm Hg below preischemic levels in Groups I and II during ischemia, whereas in Group III, intramyocardial PO2 in the circumflex region remained near preischemic levels, and in Group IV, it rose 19 mm Hg. Mean myocardial temperature during ischemia in the circumflex area was significantly higher in Group I than in Groups II, III, and IV. Peak intramyocardial PCO2 in the circumflex region was significantly less in the retrogradely perfused hearts. Retrograde coronary sinus perfusion resulted in significant improvement in recovery of LV function, uniform myocardial cooling, normal intramyocardial PO2, and less intramyocardial PCO2 accumulation, despite the presence of a total circumflex coronary artery occlusion.     

Prolonged preservation of the blood-perfused canine heart with glycolysis-promoting solution.

BACKGROUND: Prolonged ischemia and inadequate myocardial preservation remain significant perioperative risk factors in cardiac transplantation. Long-term preservation techniques that have been effective in small rodent hearts have not been as effective in larger animal models or in clinical studies. We developed a cardioplegia solution formulated to promote high-energy phosphate production from glycolysis and determined its efficacy in a blood perfused canine heart model subjected to 24 hours of ischemia. METHODS: Hearts harvested from adult dogs (n = 6 per group) were flushed with a histidine-buffered cardioplegia solution containing glucose or University of Wisconsin solution. The hearts were maintained at 4 degrees C for 24 hours then reperfused with autologous blood. After reperfusion, left ventricular pressures were measured with an intracavitary balloon at varying balloon volumes and compared with control nonischemic hearts. Predicted stroke volume and ejection fraction were calculated at an end-systolic pressure of 70 mm Hg and end-diastolic pressure of 15 mm Hg. RESULTS: Developed pressure was better preserved in the hearts that received histidine-buffered solution (93+/-9 versus 38+/-7 mm Hg, p<0.05), along with a higher end-diastolic volume at 15 mm Hg (31+/-3 versus 22+/-2 mL histidine-buffered versus University of Wisconsin solutions, respectively, p<0.05). Stroke volume and ejection fraction were also higher in the histidine group (17+/-2.5 versus 2.3+/-1.2 mL and 50%+/-3.5% versus 9% +/-4.5%, respectively) in the presence of dobutamine. CONCLUSIONS: The highly buffered glycolysis-promoting cardioplegia solution provided effective preservation of the blood perfused canine heart with superior recovery of pump performance after 24 hours of hypothermic ischemia compared with University of Wisconsin solution in this model.     

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

OBJECTIVE: delta-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 delta-opioid receptor agonist D-Ala(2)-D-Leu(5) 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 delta-opioid receptor antagonist. METHODS: Sixty-four isolated working rat hearts were subjected to 45 minutes of hypothermic ischemia at 30 degrees 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 delta-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.     

Age-related changes in the ability of hypothermia and cardioplegia to protect ischemic rabbit myocardium

Hypothermia combined with pharmacologic cardioplegia protects the globally ischemic adult heart, but this benefit may not extend to children; poor postischemic recovery of function and increased mortality may result when this method of myocardial protection is used in children. The relative susceptibilities to ischemia-induced injury modified by hypothermia alone and by hypothermia plus cardioplegia were assessed in isolated perfused immature (7- to 10-day-old) and mature (6- to 24-month-old) rabbit hearts. Hearts were perfused aerobically with Krebs-Henseleit buffer in the working mode for 30 minutes, and aortic flow was recorded. This was followed by 3 minutes of hypothermic (14 degrees C) coronary perfusion with either Krebs or St. Thomas' Hospital cardioplegic solution No. 2, followed by hypothermic (14 degrees C) global ischemia (mature hearts 2 and 4 hours; immature hearts 2, 4, and 6 hours). Hearts were reperfused for 15 minutes in the Langendorff mode and 30 minutes in the working mode, and recovery of postischemic function was measured. Hypothermia alone provided excellent protection of the ischemic immature rabbit heart, with recovery of aortic flow after 2 and 4 hours of ischemia at 97% +/- 3% and 93% +/- 4% (mean +/- standard deviation) of the preischemic value. Mature hearts protected with hypothermia alone recovered only minimally, with 22% +/- 16% recovery of preischemic aortic flow after 2 hours; none were able to generate flow at 4 hours. St. Thomas' Hospital solution No. 2 improved postischemic recovery of aortic flow after 2 hours of ischemia in mature hearts from 22% +/- 16% to 65% +/- 6% (p less than 0.05), but actually decreased postischemic aortic flow in immature hearts from 97% +/- 3% to 86% +/- 10% (p less than 0.05). To investigate any dose-dependency of this effect, we subjected hearts from both age groups to reperfusion with either Krebs solution or St. Thomas' Hospital solution No. 2 for 3 minutes every 30 minutes throughout a 2-hour period of ischemia. Reexposure to Krebs solution during ischemia did not affect postischemic function in either age group. Reexposure of immature hearts to St. Thomas' Hospital solution No. 2 caused a decremental loss of postischemic function in contrast to incremental protection with multidose cardioplegia in the mature heart. We conclude that immature rabbit hearts are significantly more tolerant of ischemic injury than mature rabbit hearts and that, unexpectedly, St. Thomas' Hospital solution No. 2 damages immature rabbit hearts.     

Adjuvant N-(2-mercaptopropionyl)-glycine in blood cardioplegia does not improve myocardial protection in ischemically damaged hearts

Oxyradicals potentially limit the myocardial protection provided by blood cardioplegia in ischemically damaged hearts. We tested the hypothesis that the addition to blood cardioplegic solution of a new oxyradical scavenger--N-(2-mercaptopropionyl)-glycine--would result in improved left ventricular performance and oxygen consumption compared to that resulting from the use of blood cardioplegia alone. Gauges and transducer-tipped catheters for left ventricular minor axis ultrasonic dimension were placed in 17 open-chest dogs, and instantaneous left ventricular pressure-diameter data were acquired by computer. The aorta was crossclamped for 30 minutes during total vented bypass to induce ischemic injury. The heart was reoxygenated and protected by multidose, hypothermic blood cardioplegic solution alone (n = 9) or enhanced with 0.0132 mmol N-(2-mercaptopropionyl)-glycine (n = 8) for 1 hour of cardioplegia-induced arrest. Preischemic and postischemic left ventricular performance was measured by slope changes in end-systolic pressure-diameter relations induced by gradual afterload reduction during right heart bypass. When blood cardioplegia alone was used, postischemic left ventricular systolic performance was depressed by 73.2% +/- 10.0% (166.8 +/- 56.1 mm Hg/mm versus 25.1 +/- 7.0 mm Hg/mm). N-(2-mercaptopropionyl)-glycine did not significantly attenuate this functional depression (62.7% +/- 9.0%, 146.6 +/- 67.6 mm Hg/mm versus 33.6 +/- 11.9 mm Hg/mm). The postischemic end-diastolic pressure-diameter relation was shifted to the right, whereas chamber stiffness was increased comparably, with or without N-(2-mercaptopropionyl)-glycine. Postischemic oxygen consumption in the beating working state, calculated from left ventricular blood flow (measured by microspheres) and arterial-coronary sinus oxygen extraction, averaged 7.8 +/- 0.9 ml O2/100 gm/min with blood cardioplegia alone and 7.5 +/- 1.0 ml O2/100 gm/min with N-(2-mercaptopropionyl)-glycine, and was unchanged from paired preischemic values in both groups. We conclude (1) that N-(2-mercaptopropionyl)-glycine added to blood cardioplegic solution in the dose and delivery regimen tested did not improve ventricular systolic and diastolic performance compared with blood cardioplegia alone and (2) that postischemic oxygen consumption may not parallel the extent of left ventricular functional recovery.     

Protection of the immature myocardium. An experimental evaluation of topical cooling, single-dose, and multiple-dose administration of St. Thomas' Hospital cardioplegic solution

Low cardiac output in infants after cardiac operations continues to be a problem, yet little experimental work has been done to evaluate the various methods of protecting the immature myocardium. In this study, we have used an isolated working heart model to test three methods of myocardial protection in 3- to 4-week-old rabbit hearts: (1) topical cooling, (2) single-dose cardioplegia plus topical cooling, and (3) multiple-dose cardioplegia plus topical cooling. Myocardial temperature was maintained at 10 degrees C during ischemia, and St. Thomas' Hospital solution was used for cardioplegia. Sets of 18 hearts were subjected to 60, 90, or 120 minutes of ischemia, and within each set six hearts were protected by all three methods. After 90 and 120 minutes of ischemia, the percent recovery of aortic flow (expressed as mean +/- standard error of the mean) was lower in hearts protected with multiple-dose cardioplegia plus topical cooling (61.5% +/- 4.8%, 50.7% +/- 14.2%) than in those protected with topical cooling (92.4% +/- 5.7%, 94.3% +/- 12.8%) or single-dose cardioplegia plus topical cooling (86.4% +/- 5.3%, 90.2 +/- 3.6%). However, adenosine triphosphate, creatine phosphate, and glycogen levels were adequately preserved in all groups. Both topical cooling and single-dose cardioplegia provide effective protection for the immature rabbit heart during ischemia, but multiple-dose cardioplegia plus topical cooling results in inadequate preservation of hemodynamic function, despite adequate preservation of myocardial high-energy phosphate stores.     

Quinaprilat during cardioplegic arrest in the rabbit to prevent ischemia-reperfusion injury

OBJECTIVES: This study evaluated intracardiac angiotensin-converting enzyme inhibition as an adjuvant to cardioplegia and examined its effects on hemodynamic, metabolic, and ultrastructural postischemic outcomes. METHODS: The experiments were performed with an isolated, erythrocyte-perfused, rabbit working-heart model. The hearts excised from 29 adult New Zealand White rabbits (2950 +/- 200 g) were randomly assigned to four groups. Two groups received quinaprilat (1 microg/mL), initiated either with cardioplegia (n = 7) or during reperfusion (n = 7). The third group received l-arginine (2 mmol/L) initiated with cardioplegia (n = 7). Eight hearts served as a control group. Forty minutes of preischemic perfusion were followed by 60 minutes of hypothermic arrest and 40 minutes of reperfusion. RESULTS: All treatments substantially improved postischemic recovery of external heart work (62% +/- 6%, 69% +/- 3%, and 64% +/- 5% in quinaprilat during cardioplegia, quinaprilat during reperfusion, and l-arginine groups, respectively, vs 35% +/- 5% in control group, P <.001) with similarly increased external stroke work and cardiac output. When administered during ischemia, quinaprilat significantly improved recovery of coronary flow (70% +/- 8%, P =.028 vs quinaprilat during reperfusion [49% +/- 5%] and P =.023 vs control [48% +/- 6%]). l-Arginine (55% +/- 7%) showed no significant effect. Postischemic myocardial oxygen consumption remained low in treatment groups (4.6 +/- 1.2 mL. min(-1). 100 g(-1), 6.0 +/- 2.2 mL. min(-1). 100 g(-1), and 4.7 +/- 1.6 mL. min(-1). 100 g(-1) in quinaprilat during cardioplegia, quinaprilat during reperfusion, and l-arginine groups, respectively, vs 4.2 +/- 0.8 mL. min(-1). 100 g(-1) in control group), even though cardiac work was markedly increased. High-energy phosphates, which were consistently elevated in all treatment groups, showed a significant increase in adenosine triphosphate with quinaprilat during ischemia (2.24 +/- 0.14 micromol/g vs 1.81 +/- 0.12 micromol/g in control group, P =.040). Ultrastructural grading of mitochondrial damage revealed best preservation with quinaprilat during ischemia (100% [no damage], P =.001 vs control). CONCLUSION: These experimental findings have clinical relevance regarding prevention of postoperative myocardial stunning and low coronary reflow in patients undergoing heart surgery.     

Bradykinin protects the rabbit heart after cardioplegic ischemia via NO-dependent pathways

Background. Depressed myocardial performance is an important clinical problem after open heart surgery. We hypothesized pretreating with bradykinin would pharmacologically precondition the heart and improve postischemic performance, and induce myocardial preconditioning by activating nitric oxide synthase.

Methods. Thirty-three rabbit hearts underwent retrograde perfusion with Krebs-Henseleit buffer (KHB) followed by 50 minutes of 37°C cardioplegic ischemia with St. Thomas’ cardioplegia solution (StTCP). Ten control hearts received no pretreatment. Ten bradykinin-pretreated hearts received a 10-minute infusion of 0.1 μMol/L bradykinin-enriched KHB and cardioplegic arrest with 0.1 μMol/L bradykinin-enriched StTCP. Six other hearts received 0.1 μMol/L HOE 140, a selective B2 receptor antagonist, added to both the 0.1 μMol/L bradykinin-enriched KHB and 0.1 μMol/L bradykinin-enriched StTCP solutions. Finally, six other hearts received 100 μMol/L of N-Ω-nitro- -arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, added to both the 0.1 μMol/L bradykinin-enriched KHB and 0.1 μMol/L bradykinin-enriched StTCP solutions.

Results. Bradykinin pretreatment significantly improved postischemic performance and coronary flow (CF) compared with control (LVDP: 53 ± 5* vs 27 ± 4 mm Hg; +dP/dt_max: 1,025 ± 93* vs 507 ± 85 mm Hg/s; CF: 31 ± 3* vs 22 ± 2 mL/min; *p < 0.05). Both HOE 140 and L-NAME abolished bradykinin-induced protection, resulting in recovery equivalent to untreated controls.

Conclusions. Bradykinin pretreatment improves recovery of ventricular and coronary vascular function via nitric oxide-dependent mechanisms. Pharmacologic preconditioning by bradykinin pretreatment may be an important new strategy for improving myocardial protection during heart surgery.     

Role Central Mu, Delta-1, and Kappa-1 Opioid Receptors in Opioid-induced Muscle Rigidity in the Rat

Background: Opioids appear to produce their physiologic effects by binding to at least three types of opioid receptors, the mu (micro), delta (delta), and kappa (kappa) receptors. Muscle rigidity occurs after administration of supra-analgesic doses of potent micro-preferring agonists like alfentanil. The role of different supraspinal opioid receptors in this rigidity has been addressed only recently. To elucidate the contribution of central micro, delta, and kappa receptors to muscle rigidity, the effects of intracerebroventricularly administered opioid receptor-selective agonists and antagonists on alfentanil-induced muscle rigidity were examined in rats.

Methods: Rats in which chronic intracerebroventricular cannulae had been implanted received an intracerebroventricular infusion of either saline or a micro (D-Ala2,N-Me-Phe4 -Gly5 -ol-enkephalin; DAMGO), delta1 (D-Pen2,D-Pen5 -enkephalin; DPDPE), or kappa1 (trans-(plus/minus)-3,4-dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohex yl)-benzene-acetamide methane sulfonate; U50,488H) opioid agonist. Ten minutes later, they received either saline or the micro-agonist alfentanil subcutaneously. Muscle rigidity was assessed using hindlimb electromyographic activity. Different groups of animals were pretreated with an intracerebroventricular infusion of either saline or a micro (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2; CTAP), delta (naltrindole), or kappa1 (norbinaltorphimine) opioid antagonist before administration of either saline or a selective intracerebroventricular agonist.

Results: The micro agonist DAMGO alone dose-dependently induced muscle rigidity. This effect was antagonized by pretreatment with the micro-selective antagonist CTAP. Neither DPDPE nor U50,488H, when administered alone, affected muscle tone. However, both the delta1 and kappa1 agonists dose-dependently attenuated alfentanil-induced rigidity. This antagonism of alfentanil rigidity was abolished after pretreatment with the delta (naltrindole) and kappa1 (nor-binaltorphimine) antagonists, respectively.     

Safety of prolonged aortic clamping with blood cardioplegia. I. Glutamate enrichment in normal hearts

This study compares the protection provided by prolonged (4 hours) aortic clamping with glutamate-enriched potassium blood cardioplegia (n = 8) to (1) prolonged (4 hours) aortic clamping with multidose potassium blood cardioplegia without glutamate (n = 4), (2) 4 hours of continuous perfusion of the beating empty heart (n = 7), and (3) 15 minutes of normothermic ischemia (n = 10). According to measurements of myocardial oxygen uptake, left ventricular compliance, left ventricular contractility, and stroke work performance, no statistical difference could be detected between those hearts receiving blood cardioplegia either with or without glutamate enrichment. In both of these groups, myocardial protection was excellent, as demonstrated by the following: postischemic myocardial oxygen uptake 43% (p less than 0.05) above control, 95% +/- 6% recovery of the left ventricular compliance, a 97% +/- 5% return of the left ventricular contractility, and a 91% +/- 6% recovery of stroke work index. Contrary to the excellent recovery of those hearts receiving blood cardioplegic protection, those hearts undergoing 4 hours of continuous perfusion showed a 45% +/- 16% (p less than 0.05) loss of left ventricular compliance and a 72% +/- 8% (p less than 0.05) recovery of stroke work index; those hearts experiencing 15 minutes of normothermic ischemia showed a 74% +/- 6% (p less than 0.05) return of left ventricular compliance, a 30% +/- 5% (p less than 0.05) decrease in contractility, and a 56% +/- 5% (p less than 0.05) recovery of postischemic left ventricular stroke work.     

Modulation of mitochondrial adenosine triphosphate-sensitive potassium channels and sodium-hydrogen exchange provide additive protection from severe ischemia-reperfusion injury

BACKGROUND: Preconditioning and inhibition of sodium-proton exchange attenuate myocardial ischemia-reperfusion injury by means of independent mechanisms that might act additively when used together. The hypothesis of this study is that treatment with a sodium-proton exchange inhibitor and a mitochondrial adenosine triphosphate-sensitive potassium channel opener produces superior functional recovery and a greater decrease in left ventricular infarct size compared with treatment with either drug alone in a model of severe global ischemia. METHODS: Isolated crystalloid-perfused rat hearts (n = 8 hearts per group) were administered vehicle (control, 0.04% dimethyl sulfoxide), diazoxide (100 micromol/L in 0.04% dimethyl sulfoxide), cariporide (10 micromol /L in 0.04% dimethyl sulfoxide), or diazoxide and cariporide before 40 minutes of ischemia at 35.5 degrees C to 36.5 degrees C and 30 minutes of reperfusion. RESULTS: The combination group had superior postischemic systolic function compared with that seen in the cariporide, diazoxide, and control groups (recovery of developed pressure: 91% +/- 7% vs 26% +/- 5%, 35% +/- 6%, and 16% +/- 3%, respectively; P <.05). Postischemic diastolic function in the combination group was superior compared with that seen in the other groups (change(pre-post) diastolic pressure of 67 +/- 4 mm Hg with control, 49 +/- 11 mm Hg with diazoxide, 59 +/- 10 mm Hg with cariporide, and 3 +/- 3 mm Hg with diazoxide and cariporide combination; P <.05). The left ventricular infarct area was less in the combination group compared with that in the cariporide, diazoxide, and control groups (6% +/- 2% vs 35% +/- 7%, 25% +/- 3%, and 37% +/- 9%, respectively; P <.05). CONCLUSIONS: Combining a selective mitochondrial adenosine triphosphate-sensitive potassium channel opener with a selective reversible inhibitor of sarcolemmal sodium-proton exchange improves recovery of contractile function from severe global ischemia in the isolated buffer-perfused rat heart. The putative mechanism for this benefit is superior protection of mitochondrial function.     

Protection of the hypertrophied pig myocardium. A comparison of crystalloid, blood, and Fluosol-DA cardioplegia during prolonged aortic clamping

The myocardial protective effects of crystalloid, blood, and Fluosol-DA-20% cardioplegia were compared by subjecting hypertrophied pig hearts to 3 hours of hypothermic (10 degrees to 15 degrees C), hyperkalemic (20 mEq/L) cardioplegic arrest and 1 hour of normothermic reperfusion. Left ventricular hypertrophy was created in piglets by banding of the ascending aorta, with increase of the left ventricular weight-body weight ratio from 3.01 +/- 0.2 gm/kg (control adult pigs) to 5.50 +/- 0.2 gm/kg (p less than 0.001). An in vivo isolated heart preparation was established in 39 grown banded pigs, which were divided into three groups to receive aerated crystalloid (oxygen tension 141 +/- 4 mm Hg), oxygenated blood (oxygen tension 584 +/- 41 mm Hg), or oxygenated Fluosol-DA-20% (oxygen tension 586 +/- 25 mm Hg) cardioplegic solutions. The use of crystalloid cardioplegia was associated with the following: a low cardioplegia-coronary sinus oxygen content difference (0.6 +/- 0.1 vol%), progressive depletion of myocardial creatine phosphate and adenosine triphosphate during cardioplegic arrest, minimal recovery of developed pressure (16% +/- 8%) and its first derivative (12% +/- 7%), and marked structural deterioration during reperfusion. Enhanced oxygen uptake during cardioplegic infusions was observed with blood cardioplegia (5.0 +/- 0.3 vol%), along with excellent preservation of high-energy phosphate stores and significantly improved postischemic left ventricular performance (developed pressure, 54% +/- 4%; first derivative of left ventricular pressure, 50% +/- 5%). The best results were obtained with Fluosol-DA-20% cardioplegia. This produced a high cardioplegia-coronary sinus oxygen content difference (5.8 +/- 0.1 vol%), effectively sustained myocardial creatine phosphate and adenosine triphosphate concentrations during the extended interval of arrest, and ensured the greatest hemodynamic recovery (developed pressure, 81% +/- 6%, first derivative of left ventricular pressure, 80% +/- 10%) and the least adverse morphologic alterations during reperfusion. It is concluded that oxygenated Fluosol-DA-20% cardioplegia is superior to oxygenated blood and especially aerated crystalloid cardioplegia in protecting the hypertrophied pig myocardium during prolonged aortic clamping.