Effects of hydrogen sulphide on ischaemia–reperfusion injury and ischaemic preconditioning in the isolated, perfused rat heart

OBJECTIVE: Hydrogen sulphide (H(2)S) protects the heart against ischaemia-reperfusion injury caused by low flow or local ischaemia. We hypothesised that: (1) H(2)S protects against global ischaemia-reperfusion injury of the heart, (2) H(2)S plays a mechanistic role in ischaemic preconditioning, and (3) H(2)S acts by phosphorylation of protein kinases. METHODS: Isolated, perfused rat hearts were used in two series. Series 1: group 1.1 (n=10), 40 min of ischaemia and 120 min of reperfusion, group 1.2 (n=7), like 1.1 except that 40 microM NaHS was added to the perfusate during stabilisation and throughout the experiment. Group 1.3 (n=10), like 1.1, but endogenously produced H(2)S was blocked by D,L-propargylglycine. Series 2: group 2.1 (n=10) control, 30 min of ischaemia followed by 120 min of reperfusion. Group 2.2 (n=10) ischaemic preconditioning before sustained ischaemia and 120 min of reperfusion. Group 2.3 (n=10) like 2.2 except of D,L-propargylglycine treatment like in group 1.3. Mitogen activated protein kinases including extracellular signal-regulated kinases (ERK 1/2), the stress-activated/c-Jun NH2 terminal kinases (JNK), P38, as well as protein kinase B/AKT (AKT), adenosine monophosphate dependent protein kinase (AMPK) and the inducible heat shock protein 72 were measured by Western blotting. Adenine nucleotides (ATP, ADP, and AMP) were measured by high-pressure liquid chromatography and energy charge was calculated. RESULTS: Infarct size was increased by D,L-propargylglycine (40+/-6 vs 27+/-10% in controls, p=0.03, Bonferroni post hoc test). There was a non-significant decrease in infarct size in the NaHS group (to 20+/-13%). Western blot analysis indicated an upregulation of heat shock protein 72 in the NaHS treated group and a reduced phosphorylation of AKT in the D,L-propargylglycine group. D,L-propargylglycine had no effect on ischaemic preconditioning or on phosphorylation of protein kinases (ERK, AKT, P38, JNK and AMPK) in preconditioned hearts. No difference in energy charge was found between groups, although ADP was increased in the NaHS-treated group. CONCLUSION: Endogenous H(2)S production protects against global ischaemia, and H(2)S may be a part of the endogenous cell defence. However, endogenous H(2)S did not appear to be important in ischaemic preconditioning, and protein kinases were not important for the effect of H(2)S. Exogenous H(2)S may provide myocardial protection, possibly acting by expression of heat shock protein 72.     

Differential Activation of Mitogen-activated Protein Kinases in Ischemic and Anesthetic Preconditioning

Background: Accumulating evidence pinpoints to the pivotal role of mitogen-activated protein kinases (MAPKs) in the signal transduction underlying cardiac preconditioning.

Methods: PD98059, an inhibitor of extracellular signal-regulated protein kinase (MEK-ERK1/2), and SB203580, an inhibitor of p38 MAPK, were used to evaluate the role of MAPKs with respect to postischemic functional recovery in isolated perfused rat hearts subjected to ischemic preconditioning (IPC) and anesthetic preconditioning (APC). Western blot analyses were used to determine the degree of ERK1/2 and p38 MAPK activation after the application of the preconditioning stimulus and after ischemia-reperfusion. Immunohistochemical staining served to visualize subcellular localization of activated MAPKs.

Results: PD98059 and SB203580 abolished postischemic functional recovery in IPC but not in APC. IPC but not APC markedly activated ERK1/2 and p38 MAPK, which were abrogated by coadministration of the specific blockers. Conversely, IPC and APC enhanced ERK1/2 activity after ischemia-reperfusion as compared to nonpreconditioned hearts, and IPC in addition enhanced p38 MAPK activity. Coadministration of PD98059 and SB203580 during IPC but not during APC inhibited postischemically enhanced MAPK activities. Moreover, chelerythrine and 5-hydroxydecanoate, effective blockers of IPC and APC, annihilated IPC- and APC-induced enhanced postischemic responses of MAPKs. Finally, administration of PD98059 during ischemia-reperfusion diminished the protective effects of IPC and APC. Immunohistochemistry revealed increased ERK1/2 activity primarily in intercalated discs and nuclei and increased p38 MAPK activity in the sarcolemma and nuclei of IPC-treated hearts.     

Differential activation of mitogen-activated protein kinases in ischemic and anesthetic preconditioning

BACKGROUND: Accumulating evidence pinpoints to the pivotal role of mitogen-activated protein kinases (MAPKs) in the signal transduction underlying cardiac preconditioning. METHODS: PD98059, an inhibitor of extracellular signal-regulated protein kinase (MEK-ERK1/2), and SB203580, an inhibitor of p38 MAPK, were used to evaluate the role of MAPKs with respect to postischemic functional recovery in isolated perfused rat hearts subjected to ischemic preconditioning (IPC) and anesthetic preconditioning (APC). Western blot analyses were used to determine the degree of ERK1/2 and p38 MAPK activation after the application of the preconditioning stimulus and after ischemia-reperfusion. Immunohistochemical staining served to visualize subcellular localization of activated MAPKs. RESULTS: PD98059 and SB203580 abolished postischemic functional recovery in IPC but not in APC. IPC but not APC markedly activated ERK1/2 and p38 MAPK, which were abrogated by coadministration of the specific blockers. Conversely, IPC and APC enhanced ERK1/2 activity after ischemia-reperfusion as compared to nonpreconditioned hearts, and IPC in addition enhanced p38 MAPK activity. Coadministration of PD98059 and SB203580 during IPC but not during APC inhibited postischemically enhanced MAPK activities. Moreover, chelerythrine and 5-hydroxydecanoate, effective blockers of IPC and APC, annihilated IPC- and APC-induced enhanced postischemic responses of MAPKs. Finally, administration of PD98059 during ischemia-reperfusion diminished the protective effects of IPC and APC. Immunohistochemistry revealed increased ERK1/2 activity primarily in intercalated discs and nuclei and increased p38 MAPK activity in the sarcolemma and nuclei of IPC-treated hearts. CONCLUSIONS: Although MAPKs may orchestrate cardioprotection as triggers and mediators in IPC, they are devoid of triggering, but they may have mediator effects in APC.     

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.     

Reduced tolerance of global ischemia in the hypertrophied heart

OBJECTIVE: Reduced coronary reserve during reperfusion may cause postischemic diastolic dysfunction in pressure-overload-induced hypertrophy. We studied the effect of coronary flow regulation (simulated hyperemic or depressed flow) on postischemic cardiac function during reperfusion. METHODS: Left ventricular pressure overload was induced in 4-week-old rats by abdominal aortic constriction. At 6 weeks of age, isolated Langendorff-perfused hearts (perfusion pressures: 75 mmHg in controls and 110 mmHg in the aortic constriction group) were subjected to hypothermic global ischemia (15 degrees C, 210 min), followed by 2 types of coronary flow regulation during the initial 20 min of reperfusion--manipulated high flow in control hearts (group I), manipulated low flow in control hearts (group II), manipulated high flow in aortic constriction hearts (group III), and manipulated low flow in aortic constriction hearts (group IV) (n = 6/group), and then constant pressure perfusion during the subsequent 45 min of reperfusion. Cardiac function was measured using an isovolumic balloon in the pre- and postischemic periods. RESULTS: Aortic constriction hearts exhibited greater left ventricular end-diastolic pressure than did control hearts. The increase in left ventricular end-diastolic pressure did not differ between group I (3 +/- 2 mmHg) and group II (-1 +/- 1 mmHg) or between group III (29 +/- 5 mmHg) and group IV (30 +/- 6 mmHg). No difference was seen in postischemic recovery of left ventricular systolic pressure between high and low flow groups in control and aortic constriction hearts. CONCLUSION: Manipulations in coronary flow during reperfusion did not affect postischemic cardiac function in control or aortic constriction hearts, suggesting that depressed coronary flow during early reperfusion is not a primary cause of postischemic diastolic dysfunction in the hypertrophied myocardium.     

普萘洛尔预处理对大鼠肥厚心肌缺血再灌注损伤的保护作用及其机制探讨

目的　探讨普萘洛尔预处理对肥厚心肌缺血再灌注损伤作用的影响及机制。方法　 36只雄性 8周龄SD大鼠 ,随机分为正常心肌组、肥厚心肌对照组及普萘洛尔组。腹主动脉缩窄制备大鼠心肌肥厚模型 ,围手术期应用普萘洛尔预处理 ,采用离体工作心脏灌流模型 ,观察心功能指标 ,并取心肌组织行形态学定量测定线粒体损伤程度。结果　围手术期应用普萘洛尔预处理 ,可明显改善肥厚心肌缺血再灌注后心功能的恢复 ,增加冠状动脉流量 ,且心肌细胞线粒体形态学定量测定显示 ,普萘洛尔预处理组心肌细胞线粒体损伤较对照组明显减轻 (P <0 0 1)。结论　普萘洛尔预处理对大鼠肥厚心肌缺血再灌注损伤具保护作用 ,其机制可能与减轻心肌线粒体损伤 ,明显保护再灌注期间线粒体良好呼吸功能有关。     

Opioid preconditioning: myocardial function and energy metabolism.

BACKGROUND: Opioid receptor agonists are involved in ischemic preconditioning and natural hibernation. The aim of this study was to determine whether pretreatment with D-Ala2-Leu5-enkephalin or morphine confers cardioprotection in large mammalian hearts. We assessed myocardial functional recovery and global energy metabolism after ischemic cold storage. METHODS: After pretreatment with D-Ala2-Leu5-enkephalin, morphine sulfate, or saline (n = 6 each), swine hearts were excised and stored for 75 minutes at 4 degrees C, then reperfused in a four-chamber isolated working heart apparatus. Serial myocardial biopsies were performed to assess cellular energy metabolism. RESULTS: Improved systolic (cardiac output, contractility) and diastolic (tau) left ventricular functions were observed in hearts pretreated with D-Ala2-Leu5-enkephalin or morphine. These benefits were not correlated with changes in high-energy phosphate levels. Cardiac enzyme leakage (creatine kinase, troponin-I) was similar among treated and control groups. Lactate efflux increased significantly in controls, but not in opioid-pretreated hearts (p < 0.01) at 75 minutes of reperfusion. CONCLUSIONS: D-Ala2-Leu5-enkephalin and morphine pretreatments improve postischemic function after cold storage of swine hearts. Postischemic lactate reduction, but not high-energy phosphate levels, may account for the observed cardioprotective effects.     

Effect of carnitine on myocardial function and metabolism following global ischemia

Carnitine has therapeutic potential for the postischemic heart by facilitating the oxidation of acylated fatty acid metabolites, the intracellular accumulation of which has a deleterious effect on myocardial function and metabolism. To test this hypothesis, two groups of dogs were given preischemic treatment with carnitine, 50 mg per kilogram of body weight (Group 1) or 100 mg/kg (Group 2), and were compared with untreated controls (N = 12 for all groups). The canine hearts underwent 30 minutes of global 37 degrees C ischemic arrest with reperfusion. Left ventricular systolic and diastolic function was assessed by an intracavitary balloon while metabolic derangements were quantitated by serial myocardial biopsies assayed for adenosine triphosphate (ATP). Comparable 49 to 53% (p less than 0.01) declines in preischemic ATP levels occurred during the study period in the controls and both experimental groups. However, postischemic systolic left ventricular function was better preserved in Group 2: these hearts generated 61 +/- 3% of preischemic peak developed pressure compared with 37 +/- 4% in the controls and 42 +/- 3% in Group 1 (p less than 0.01 for each), and 60 +/- 2% of preischemic maximum rate of rise of left ventricular pressure as opposed to 45 +/- 4% in the controls and 49 +/- 6% in Group 1 (p less than 0.02 for each).(ABSTRACT TRUNCATED AT 250 WORDS)     

Left ventricular mechanical unloading restores Beta-2-adrenergic receptor mRNA expression and decreases susceptibility to ischemia and reperfusion in the failing heart

OBJECTIVES: Left ventricular (LV) mechanical unloading is known to reduce the hemodynamic demands of failing LV, resulting in improved myocyte contractility. This study was designed to examine effects of LV unloading on beta-adrenergic receptor (BAR) expression and ischemic susceptibility to ischemia reperfusion. METHODS: Five groups were studied: group 1 [unloading myocardial infarction (MI), n = 6], MI hearts 2 weeks after coronary ligation subjected to LV unloading by heterotopic heart transplantation for 2 weeks; group 2 (2-week MI, n = 6), MI hearts left for 2 weeks without unloading; group 3 (4-week MI, n = 6), MI hearts left for 4 weeks without unloading; group 4 (control, n = 6), normal (non-MI) hearts as a control with no interventions, and group 5 (unloading control, n = 5), normal (non-MI) hearts subjected to LV unloading for 2 weeks. Then, all hearts were isolated and subjected to 20 min of global ischemia and 60 min of reperfusion on Langendorff apparatus. LV pressures and coronary flow were measured throughout the experiment. Either total BAR density or beta(2)-adrenergic receptor (B2AR) mRNA expression in the noninfarcted myocardium was determined by radioligand binding assays or real-time quantitative RT-PCR, respectively. RESULTS: LV unloading improved postischemic functional recovery (unloading MI vs. 2-week MI vs. 4-week MI: 74 +/- 6 vs. 54 +/- 5 vs. 51 +/- 4%; p < 0.05 vs. unloading MI). LV unloading restored B2AR mRNA expression (unloading MI vs. 2-week MI vs. 4-week MI: 4.78 +/- 0.21 vs. 2.80 +/- 0.19 vs. 2.24 +/- 0.17 x 10(7) copy/microg total RNA; p < 0.05 vs. unloading MI). CONCLUSION: LV mechanical unloading restored B2AR mRNA expression and improved postischemic functional recovery.     

Amplification by hyperoxia of coronary vasodilation induced by propofol

We tested the hypothesis that in vitro coronary and myocardial effects of propofol (10-300 microM) should be significantly modified in an isolated and erythrocyte-perfused rabbit heart model in the absence (PaO(2) = 137 +/- 16 mm Hg, n = 12) or in the presence (PaO(2) = 541 +/- 138 mm Hg, n = 12) of hyperoxia. The induction of hyperoxia provoked a significant coronary vasoconstriction (-13% +/- 7%). Propofol induced increased coronary vasodilation in the presence of hyperoxia. Because high oxygen tension has been reported to induce a coronary vasoconstriction mediated by the closure of adenosine triphosphate-sensitive potassium channels, we studied the effects of propofol in 2 additional groups of hearts (n = 6 in each group) pretreated by glibenclamide (0.6 microM) and cromakalim (0.5 microM) in the absence and presence of hyperoxia, respectively. The pretreatment by glibenclamide induced a coronary vasoconstriction (-16% +/- 7%) which did not affect propofol coronary vasodilation. The pretreatment by cromakalim abolished the amplification of propofol coronary vasodilation in the presence of hyperoxia. Propofol induced a significant decrease in myocardial performance for a concentration >100 micro M both in the absence and presence of hyperoxia. We conclude that propofol coronary vasodilation is amplified in the presence of hyperoxia. This phenomenon is not explained by the previous coronary vasoconstriction induced by glibenclamide. However, the pretreatment of hearts by cromakalim abolished the amplification of propofol coronary vasodilation in the presence of hyperoxia. The myocardial effects of propofol were not affected by the presence of hyperoxia. IMPLICATIONS: Propofol induced a coronary vasodilation that was amplified in the presence of hyperoxia. This phenomenon does not seem to be related to previous coronary vasoconstriction. The myocardial effects of propofol were not significantly modified in the presence of hyperoxia.     

Nicardipine: myocardial protection in isolated working hearts.

The effectiveness of the calcium antagonist nicardipine in protecting the ischemic myocardium was evaluated using the hemodynamic recovery of isolated working rat hearts subjected to hyperkalemic cardiac arrest followed by ischemia at 37.5 degrees C and 10 degrees C. Rat hearts (n = 51) received 20 mL of cardioplegia and were subjected to 27 minutes of ischemia at 37.5 degrees C. Group A (control) did not receive nicardipine. Groups B through F received nicardipine in the cardioplegia with total doses ranging from 2 micrograms to 6 micrograms. Group A had 46% survival of ischemia, whereas groups C (3 micrograms) and D (4 micrograms) had survival rates of 88% and 100%, respectively (p less than 0.05). The recovery of aortic flow after ischemia was 35% in group A, compared with 76% in group B (2 micrograms) and 81% in group D (p less than 0.05). Group A had 49% postischemic recovery of cardiac output, whereas groups B and D had 82% and 85% recovery (p less than 0.05). The postischemic recovery of stroke volume was 48% in group A compared with 84% in group B, 87% in group D, and 73% in group E (5 micrograms) (p less than 0.05). Additional rats were exposed to 210 minutes of ischemia (n = 41) or 240 minutes of ischemia (n = 56) at 10 degrees C. Control groups did not receive nicardipine, whereas treatment groups received nicardipine in the cardioplegia with total doses ranging from 1.4 micrograms to 6.4 micrograms. There were no significant differences in the survival of ischemia or the recovery of function after ischemia at 10 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acute severe postischemic myocardial depression reversed by triiodothyronine.

The purpose of this study was to determine the effects of triiodothyronine (T3) on postischemic left ventricular performance and high-energy phosphate content in a severe injury model. Isolated working rat hearts (n = 63) received 20 mL of hyperkalemic NIH No. 1 cardioplegia and were subjected to 20 minutes of ischemia at 37 degrees C. Treated hearts were reperfused with T3-supplemented modified Krebs-Henseleit buffer. Control hearts did not receive T3 supplementation. All treated hearts (n = 44) performed work after ischemia, whereas 26% (5/19) of the control hearts were not able to perform any left ventricular work after ischemia. Comparisons with preischemic values demonstrated significant progressive hemodynamic recovery with increasing concentrations of T3 (0, 0.06, 0.15, and 0.60 ng/mL) with concomitant recovery of left ventricular stroke work index (63%, 72%, 89% [p less than 0.05], and 99% [p less than 0.05], respectively). There were corresponding increases in recovery of aortic flow, systolic pressure, cardiac index, and stroke volume index (p less than 0.05). There were no significant changes in coronary sinus flow or heart rate in any group compared with preischemic values. Comparisons of postischemic high-energy phosphate concentrations also demonstrated no change between treated and untreated groups (p greater than 0.05). We conclude that administration of T3 in a severe left ventricular injury model significantly augments rapid ventricular recovery with no change in postischemic high-energy phosphate concentrations.     

Prevention of free radical-induced myocardial injury by allopurinol. Experimental study in cardiac preservation and transplantation

To determine the role of free radical-induced injury during heart preservation and transplantation, we harvested hearts from 28 mongrel dogs (12.5 to 16.5 kg), divided them into four groups, and orthotopically transplanted them. A group of seven hearts were orthotopically transplanted immediately after excision (group A). A second group of seven animals received allopurinol pretreatment (50 mg/kg/day) for 72 hours, and the hearts were orthotopically transplanted immediately after excision (group B). A third group of seven hearts were transplanted after continuous perfusion with oxygenated modified Collins solutions at 4 degrees C, pH 7.4, and a pressure of 20 mm Hg for 18 hours (group C). A fourth group of seven animals received allopurinol pretreatment (50 mg/kg/day) for 72 hours, and the hearts were orthotopically transplanted after perfusion with modified Collins solutions in the same manner as group C hearts (group D). The generation of free radicals, estimated by measurement of thiobarbituric acid reactive substances (malondialdehyde) in the coronary effluent, stayed at low levels during perfusion in groups C and D and also remained at low levels during operational ischemia in group A and B. During reperfusion, their levels abruptly and significantly increased and were associated with a corresponding increase in creatinine kinase MB isoenzyme (malondialdehyde levels at 30 minutes' reperfusion: A, 2.25 +/- 0.43; B, 1.55 +/- 0.25 nmol/ml/100 gm wet weight [p less than 0.05 versus group A]; C, 2.67 +/- 0.28; D, 1.77 +/- 0.27 nmol/ml/100 gm wet weight [p less than 0.05 versus group C]). In the allopurinol pretreatment groups, allopurinol significantly slowed the appearance of malondialdehyde and the release of creatinine kinase MB isoenzyme during reperfusion. Furthermore, cardiac functions during reperfusion, expressed as percent of control (mean +/- standard deviation), were significantly better in the allopurinol pretreatment groups than in the untreated groups: maximum first derivative of left ventricular pressure: A, 76.4 +/- 9.5; B, 99.7 +/- 14.3 [p less than 0.05 versus group A]; C, 25.2 +/- 2.6; D, 42.7 +/- 7.9 [p less than 0.05 versus group C]). These results indicate that (1) the generation of oxygen free radical is not significant during perfusion with modified Collins solutions nor during operational ischemia, but only during reperfusion, and (2) allopurinol reduces free radical-induced injury during reperfusion. Allopurinol has potential application in the prevention of reperfusion injury during heart transplantation.     

Preconditioning protects the severely atherosclerotic mouse heart

BACKGROUND: Coronary atherosclerosis has profound effects on vascular and myocardial biology, and it has been speculated that the atherosclerotic heart does not benefit from ischemic preconditioning. METHODS: To investigate if atherosclerosis would influence the preconditioning response, Apolipoprotein E/low density lipoprotein (LDL) receptor double knockout mice (ApoE/LDLr-/-) were fed an atherogenic diet (21% fat, 0.15% cholesterol) for 6 to 8 months. At that time, extensive atherosclerotic lesions throughout the coronary tree were seen in transverse sections stained with Oil Red-O. Hearts of ApoE/LDLr-/- mice were Langendorff-perfused with 40 minutes of global ischemia and 60 minutes reperfusion, and compared with C57BL/6 controls. Preconditioning with two episodes of 2 minutes of ischemia and 5 minutes reperfusion, or exposing the mice to a hyperoxic environment (O2 > 98%) for 60 minutes before heart perfusion, was performed. RESULTS: Hearts of mice with coronary atherosclerosis had worse postischemic function, and increased infarct size and troponin T release compared to hearts of C57BL/6 mice. Ischemic preconditioning improved postischemic ventricular function, and reduced myocardial infarct size and troponin T release in both normal and ApoE/LDLr-/- mice. The effects were most pronounced in ApoE/LDLr-/- hearts. Exposure to hyperoxia exerted a similar protection of function and cell viability of ApoE/LDLr-/- mice hearts. CONCLUSIONS: These findings suggest that the severely atherosclerotic heart may be protected by preconditioning induced by ischemia or hyperoxia.     

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.     

Improved myocardial preservation with short hyperthermia prior to cold cardioplegic ischemia in immature rabbit hearts.

OBJECTIVE: Recent observations have been shown that the induction and accumulation of heat shock proteins (HSPs) by short exposure to nonlethal whole-body hyperthermia with normothermic recovery are closely associated with transient resistance to subsequent ischemia-reperfusion challanges. Here, this study was performed to investigate whether a shortly heat shock pretreatment affects the left ventricular (LV) function after cold cardioplegic ischemia in reperfused neonatal rabbit hearts. METHODS: Hearts from neonatal New Zealand White rabbits were isolated perfused (working heart preparation) and exposed to 2 h of cold cardioplegic ischemia followed by reperfusion for 60 min. To induce the heat shock response neonatal rabbits (n=5, HT-group) were subjected to whole-body hyperthermia at 42.0-42.5 degrees C for 15 min, followed by a normothermic recovery period of 60 min, before harvesting and the onset of global hypothermic cardioplegic arrest. Another set of hearts (n=5, control group) without a heat treatment underwent a similar perfusion and ischemia protocol served as control. The postischemic recovery was assessed by measuring several parameters of LV function. LV biopsies from all control and heat treated animals were taken before ischemia and at the end of reperfusion to examine myocardial HSP levels by Western blot analysis. RESULTS: At 60 min of reperfusion the HT-group showed significant better recovery of ventricular function such as LV developed pressure (DP) (74.6+/-10 vs. 52.1+/-8.5%, P<0.05), LV positive dP/dt (910+/-170 vs. 530+/-58 mmHg/s, P<0.01) and LV end-diastolic pressure (LVEDP) (8+/-2 vs. 18.4+/-5 mmHg, P<0.05) than control. Myocardial oxygen consumption (MVO(2)) was significantly higher in the HT-group compared with control (0.054+/-0.006 vs. 0.041+/-0.002 ml/g per min, P<0.05). Significant postreperfusion lower level in lactate production was observed in the HT-group (0.83+/-0.11 vs. 1.67+/-0.8 mmol/l, P<0.05). Also, the recovery of hemodynamic parameters such as aortic flow, coronary flow and cardiac output was significantly superior (P<0.05) in the HT-group. Furthermore, high expression of HSP72(+)/73(+) were detected in the myocardial tissue samples of heat-treated rabbits by immunoblotting, appearing even at 60 min of normothermic recovery after heat stress. CONCLUSIONS: These data in the immature rabbit heart indicate that previous shortly heat treatment with high level expression of heat shock proteins (HSP72(+)/73(+)) before hypothermic cardioplegic ischemia provides transient tolerance against myocardial injury and could be an improvement for the postischemic functional recovery of neonatal hearts.     

Activation of pulmonary mitogen-activated protein kinases during cardiopulmonary bypass

PURPOSE: Cardiopulmonary bypass (CPB) produces an inflammatory response associated with pulmonary dysfunction. Mitogen-activated protein kinases (MAPK) have been shown to mediate pulmonary injury. We hypothesized that MAPK are activated during CPB and potentially contribute to lung injury. METHODS: Pigs were placed on CPB (n = 6) for 90 min, which included 80 min of cardioplegic arrest, followed by 180 min of post-CPB reperfusion. Control animals (n = 6) underwent sternotomy and heparinization only. Lung samples were collected at baseline, during CPB, and during post-CPB reperfusion. Activated forms of extracellular signal-regulated kinases 1/2 (ERK1/2) and p38 were measured by Western blot. Immunohistochemistry was used for tissue localization of activated MAPK. Pulmonary inflammation was determined by histology. Pulmonary edema was estimated by tissue water percentage. RESULTS: Activated ERK1/2 and p38 were increased after 90 min of CPB compared with controls (3.94 +/- 0.61- and 2.49 +/- 0.15-fold increase, respectively; both P < 0.01). At 180 min of post-CPB reperfusion, ERK1/2 activity was increased by nearly 5-fold compared with controls (P < 0.01), whereas p38 activity returned to baseline levels. By immunohistochemistry, activated ERK1/2 and p38 in the CPB group were localized to alveolar epithelial cells, vascular endothelial cells, and bronchial smooth muscle. Histologic signs of lung injury included leukocyte infiltration in the CPB group. Tissue water percentage was increased with CPB (89.9 +/- 1.5% versus 82.5 +/- 1.0%, CPB versus control, P < 0.05). CONCLUSIONS: The results of our study demonstrate that CPB increases pulmonary p38 activity and causes sustained activation of ERK1/2. MAPK activation thus may in part mediate the pulmonary inflammatory response and provide a potential site of intervention to prevent pulmonary dysfunction due to CPB.     

Effects of inhibition of myocardial extracellular-responsive kinase and P38 mitogen-activated protein kinase on mechanical function of rat hearts after prolonged hypothermic ischemia

BACKGROUND: Mitogen-activated protein kinases (MAPKs), including extracellular-responsive kinase (ERK) and p38 MAPK, are activated by stresses associated with hypothermia-rewarming and ischemia-reperfusion. Their activation in heart is associated with beneficial (preconditioning) and adverse effects (apoptosis and impaired contractility). This study determined whether ERK and p38 MAPK activities are altered by hypothermic ischemia and normothermic reperfusion and the consequences of their inhibition on recovery of myocardial function. METHODS: Left ventricular work (L x min(-1) x mm Hg) was assessed during normothermic perfusion (30 min) of isolated rat hearts that were either freshly excised or previously subjected to hypothermic storage (8 hr, 3 degrees C) and rewarming (10 min, 37 degrees C) before normothermic reperfusion (30 min). Phospho-specific immunoblot analysis of p38 MAPK was performed in hearts and various cultured cells. RESULTS: Compared with fresh hearts, hearts subjected to hypothermia and rewarming demonstrated impaired left ventricular work (1.96+/-0.53, n=12 vs. 8.37+/-0.46, n=4, <0.05) during reperfusion. The ERK inhibitor, PD98059 (20 microM), present during storage and rewarming, caused modest improvement (3.66+/-0.75, n=9, <0.05). The p38 MAPK inhibitor, SB202190 (10 microM), when present during reperfusion, improved recovery (to 6.12+/-0.75, n=6, <0.05); it was ineffective if present only during rewarming (1.52+/-0.88, n=4). In rat2 fibroblasts, hypothermia and rewarming activated p38 MAPK and its downstream kinase MAPK-activated protein kinase 2, but not c-Jun N-terminal kinase/stress-activated protein kinase. CONCLUSIONS: Myocardial p38 MAPK and MAPK-activated protein kinase 2 are stimulated by hypothermia, ischemia, and rewarming and are detrimental to recovery of mechanical function of hearts subjected to prolonged hypothermic storage. Inhibition of p38 MAPK may be useful in protocols to improve the recovery of mechanical function of cold-stored hearts.     

The role of magnesium in postischemic cardiac dysfunction.

BACKGROUND. The biochemical basis for postischemic myocardial stunning is not fully elucidated. Magnesium is an important regulator of cellular energetic processes and excitation-contraction coupling. We hypothesized that the decrease in function in the postischemic period may be the result of an alteration in magnesium regulation. METHODS. In a Langendorf perfused rabbit heart model, we used 31P nuclear magnetic resonance spectroscopy to noninvasively determine intracellular Mg2+ and high-energy phosphate levels in the preischemic period and after a 30-minute period of normothermic ischemia. We measured adenosine triphosphate (ATP), phosphocreatine, and the phosphocreatine/inorganic phosphate ratio and calculated the free energy of ATP hydrolysis (delta GATP). On reperfusion, hearts were divided into three groups (n = 7 per group)--those receiving unmodified Krebs-Henseleit (control), 192 ng/ml dobutamine, or 5 mmol/L pyruvate. RESULTS. Function (expressed as the rate-pressure product) was approximately 77% of preischemic values in the control group, whereas in both dobutamine and pyruvate groups it returned to preischemic levels. ATP was decreased similarly in all groups in the postischemic period. Phosphocreatine/inorganic phosphate ratio and delta GATP were higher in the pyruvate group compared with the other groups. Intracellular Mg2+ was elevated significantly in the unmodified control postischemic group compared with preischemic, postischemic dobutamine, and pyruvate groups (1.0 +/- 0.12 vs 0.80 +/- 0.08, 0.64 +/- 0.08, and 0.70 +/- 0.05 mmol/L, respectively; p less than 0.05). CONCLUSIONS. We conclude that (1) postischemic "stunned" hearts have elevated Mg2+ levels in association with impaired contractile function, (2) inotropic agents improve contractile function in association with a decline in Mg2+ to preischemic levels despite differing effects on intracellular energetics, and (3) Mg2+ may play an important regulatory role in the heart after ischemia.     

Effect of ischemic pretreatment on heat shock protein 72, neurologic outcome, and histopathologic outcome in a rabbit model of spinal cord ischemia

OBJECTIVE: In the present study, we investigated the effect of ischemic pretreatment on heat shock protein 72 concentration and neurologic and histopathologic outcome after transient spinal cord ischemia. METHODS: In 28 New Zealand White rabbits, an aortic occlusion device was placed infrarenally. The animals were randomly assigned to 2 groups: ischemic pretreatment (n = 14 animals) and control (n = 14 animals). The duration of ischemic pretreatment was 6 minutes. After 24 hours, the aorta was occluded for 26 minutes in both groups of animals. Neurologic function was assessed 24 and 48 hours after the definite ischemic insult. At 48 hours, the animals were killed for histopathologic evaluation of the spinal cord. In a separate set of animals, heat shock protein 72 levels were determined in the lumbar spinal cord after both a 6- and 10-minute ischemic period, with the use of a Western blot analysis. RESULTS: No significant difference in neurologic outcome between the groups was observed at 24 and 48 hours. The incidence of paraplegia and severe paresis at 48 hours was 79% in the control group and 92% in the ischemic pretreatment group. There was no difference in histopathologic scores between the groups. Heat shock protein 72 could be clearly detected 1 and 2 days after 6- or 10-minute periods of spinal cord ischemia. CONCLUSIONS: In the present rabbit study, ischemic pretreatment could not induce tolerance against a moderately severe spinal cord ischemic insult, despite increased heat shock protein 72 levels after the preconditioning stimulus.