Intracellular Catalase Inhibition Does not Predispose Rat Heart to Ischemia-Reperfusion and Hydrogen Peroxide-Induced Injuries

The objective of this study was to determine whether inhibition of intracellular catalase would decrease the tolerance of the heart to ischemia-reperfusion and hydrogen peroxide-induced injuries. Isolated bicarbonate buffer-perfused rat hearts were used in the study. Intracellular catalase was inhibited with 3-amino-1,2,4-triazole (ATZ, 1.5 g/kg body weight, two hours prior to heart perfusion). In the ischemia-reperfusion protocol, hearts were arrested with St. Thomas' II cardioplegic solution, made ischemic for 35 min at 37°C, and reperfused with Krebs-Henseleit buffer for 30 min. The extent of ischemic injury was assessed using postischemic contractile recovery and lactate dehydrogenase (LDH) leakage into reperfusate. In the hydrogen peroxide infusion protocol, hearts were perfused with increasing concentrations of hydrogen peroxide (inflow rates 0.05-1.25 μmol/min). Inhibition of catalase activity (30.4 ± 1.8 mU/mg protein in control vs 2.4 ± 0.3 mU/mg in ATZ-treated hearts) affected neither pre-ischemic aerobic cardiac function nor post-ischemic functional recovery and LDH release in hearts subjected to 35 min cardioplegic ischemic arrest. Myocardial contents of lipid hydroperoxides were similar in control and ATZ-treated animals after 20 min aerobic perfusion, ischemia, and ischemia-reperfusion. During hydrogen peroxide perfusion, there was an increase in coronary flow rate followed by an elevation in diastolic pressure and inhibition of contractile function in comparison with control hearts. The functional parameters between control and ATZ-treated groups remained unchanged. The concentrations of myocardial lipid hydroperoxides were the same in both groups. We conclude that inhibition of myocardial catalase activity with ATZ does not predispose the rat heart to ischemia-reperfusion and hydrogen peroxide-induced injury.     

SEQUENTIAL ELECTROCARDIOGRAPHIC ANALYSIS OF STONE HEART SYNDROME IN A PATIENT SUPPORTED SIX DAYS WITH AN ABDOMINAL LEFT VENTRICULAR ASSIST DEVICE (ALVAD)

A 21-year-old male patient underwent aortic and mitral valve replacement for progressive cardiac failure due to acute bacterial endocarditis. Ischemic myocardial contracture developed during attempts to restore cardiac activity following hypothermic, ischemic, cardioplegic arrest. An abdominal left ventricular assist device (ALVAD) was implanted and supported the circulation for nearly six days prior to cardiac transplantation. The preoperative EKG showed sinus tachycardia with left anterior hemiblock. Postoperatively, there was complete electromechanical dissociation. The postoperative EKG showed a superior and leftward shift of the axis. There was a marked loss of QRS voltage and variable degrees of atrioventricular block. At times, only P waves were present. On the fourth postoperative day, there was an axis shift to the extreme right. Prior to transplantation, sinus rhythm returned, and the axis shifted leftward once again. The common denominator of all the abnormal postoperative electrocardiograms was the conspicuous low voltage that probably signified early and extensive myocardial damage. To our knowledge, this is the first instance wherein a sequential electrocardiographic analysis of stone heart syndrome has been undertaken.     

Is there an effect of ischemic conditioning on myocardial contractile function following acute myocardial ischemia/reperfusion injury?

Ischemic conditioning induces cardioprotection; the final infarct size following a myocardial ischemic event is reduced. However, whether ischemic conditioning has long-term beneficial effects on myocardial contractile function following such an ischemic event needs further elucidation. To date, ex vivo studies have shown that ischemic conditioning improves the contractile recovery of isolated ventricular papillary muscle or atrial trabeculae following simulated ischemia. However, in vivo animal studies and studies in patients undergoing elective cardiac surgery show conflicting results. At the subcellular level, it is known that ischemic conditioning improved energy metabolism, preserved mitochondrial respiration, ATP production, and Ca²⁺ homeostasis in isolated mitochondria from the myocardium. Ischemic conditioning also presents with post-translational modifications of proteins in the contractile machinery of the myocardium. The beneficial effects on myocardial contractile function need further elucidation. This article is part of a Special Issue entitled: The power of metabolism: Linking energy supply and demand to contractile function edited by Torsten Doenst, Michael Schwarzer and Christine Des Rosiers.     

Effect of superoxide dismutase and catalase on myocardial energy metabolism during ischemia and reperfusion

Survival of cardiac patients undergoing heart surgery depends critically upon the recovery of myocardial energy metabolism during reperfusion of ischemic myocardium. The present study compares various parameters of myocardial energy metabolism using an isolated in situ pig heart. The left anterior descending (LAD) coronary artery was occluded for 60 min, followed by 60 min of global hypothermic cardioplegic arrest and 60 min of reperfusion. Free radical scavengers [superoxide dismutase SOD and catalase] were used to protect the ischemic heart from reperfusion injury. In both control and SOD plus catalase-treated groups, ATP, creatine phosphate (CP), ATP/ADP ratio, energy charge and phosphorylation potential dropped significantly during ischemic insult. After reperfusion, CP, ATP/ADP ratio and phosphorylation potential improved significantly, but they were restored to control level only in treated animals. In either case, free energy of ATP hydrolysis (delta G) lowered only by 5% during ischemia, but recovered promptly upon reperfusion. SOD and catalase also improved coronary blood flow and reduced creatine kinase release compared to those of untreated animals, suggesting improved myocardial recovery upon reperfusion. Our results suggest that SOD and catalase significantly improve the myocardial recovery during reperfusion by enhancing rephosphorylation steps, and the value of delta G is more critical compared to those of ATP and CP for myocardial recovery.     

Sustained postischemic cardiodepression following magnesium-diltiazem cardioplegia

Magnesium-diltiazem cardioplegia was evaluated in the intact, perfused rat heart to determine whether the joint administration of these agents would adversely affect myocardial contractile and high-energy phosphate recovery following intermittent, normothermic global ischemic arrest. Sequential metabolic and functional analyses were performed on isolated perfused rat hearts during each phase of the experimental protocol: control (10 min), normoxic cardioplegia (10 min), intermittent global ischemic arrest (two 15-min periods separated by 2 min infusion of the normoxic cardioplegic perfusate), and normoxic postischemic control reperfusion (60 min). Four different cardioplegic solutions were evaluated: 30 mM KCl, 30 mM KCl with 2 mg diltiazem/liter, 20 mM MgCl2, and 20 mM MgCl2 with 2 mg diltiazem/liter. Myocardial phosphatic metabolite levels and intracellular pH were analyzed nondestructively in the intact hearts by phosphorus-31 NMR spectroscopy. Corresponding measurements of peak left intraventricular pressure, rate of peak pressure development (dP/dt), and contraction frequency were performed at the midpoint during each 5-min interval of 31P NMR signal averaging. Magnesium plus diltiazem-treated hearts were distinguished from all other groups by a marked delay in postischemic functional recovery consisting of a prolonged depression in contractility (34% of control, P less than 0.01) that persisted throughout the first 50 min of postischemic reperfusion. Diltiazem in combination with magnesium cardioplegia was detrimental to postischemic functional recovery, despite a rapid restoration of high-energy phosphate stores. The apparent adverse interactive effects of excess magnesium and diltiazem suggest that elective ischemic arrest with magnesium cardioplegia in combination with diltiazem may be contraindicated clinically. The mechanistic basis and drug specificity of this response require further clarification. The present findings appear to exclude ATP and PCr production, and structural causes as the basis for the observed aberrant functional recovery from global ischemia of magnesium plus diltiazem-arrested hearts.     

Effectiveness of protecting the myocardium against ischemia with a normothermic cardioplegic solution and creatine phosphate

The protective effects of cardioplegic solutions (CS) containing creatine phosphate (CP) were studied in a rat heart model of cardiopulmonary bypass and ischemic cardiac arrest. Isolated rat hearts were subjected to a 3-minute coronary infusion with CS containing CP in normothermic (37 degrees C) and hypothermic (4-6 degrees C) regimes. In the normothermia group, the postischemic functional recovery was 70-75% of the preischemic control value, while the cellular ATP and CP content was reduced but insignificantly. By contrast, in the hypothermia group, the postischemic functional recovery was markedly depressed, with the tissue high-energy phosphate content being appreciably lowered. The data obtained confirm high efficacy of CP-containing cardioplegic solutions administered under normothermia conditions.     

Energy metabolism and mechanical recovery after cardioplegia in moderately hypertrophiedrats

It is well established that severe hypertrophy induces metabolic and structural changes in the heart which result in enhanced susceptibility to ischemic damage during cardioplegic arrest while much less is known about the effect of cardioplegic arrest on moderately hypertrophied hearts. The aim of this study was to elucidate the differences in myocardial high energy phosphate metabolism and in functional recovery after cardioplegic arrest and ischemia in mildly hypertrophied hearts, before any metabolic alterations could be shown under baseline conditions.Cardiac hypertrophy was induced in rats by constriction of the abdominal aorta resulting in 20% increase in heart weight/body weight ratio (hypertrophy group) while sham operated animals served as control. In both groups, isolated hearts were perfused under normoxic conditions for 40 min followed by infusion of St.Thomas' Hospital No. 1 cardioplegia and 90 min ischemia at 25øC with infusions of cardioplegia every 30 min. The changes in ATP, phosphocreatine (PCr) and inorganic phosphate (Pi) were followed by³¹ P nuclear magnetic resonance (NMR) spectroscopy. Systolic and diastolic function was assessed with an intraventricular balloon before and after ischemia.Baseline concentrations of PCr, ATP and Pi as well as coronary flow and cardiac function were not different between the two groups. However, after cardioplegic arrest PCr concentration increased to 61.8 ± 4.9 mol/g dry wt in the control group and to 46.3 ± 2.8 mol/g in hypertrophied hearts. Subsequently PCr, pH and ATP decreased gradually, concomitant with an accumulation of Pi in both groups. PCr was transiently restored during each infusion of cardioplegic solution while Pi decreased. PCr decreased faster after cardioplegic infusions in hypertrophied hearts. The most significant difference was observed during reperfusion: PCr recovered to its pre-ischemic levels within 2 min following restoration of coronary flow in the control group while similar recovery was observed after 4 min in the hypertrophied hearts. A greater deterioration of diastolic function was observed in hypertrophied hearts.Moderate hypertrophy, despite absence of metabolic changes under baseline conditions could lead to enhanced functional deterioration after cardioplegic arrest and ischemia. Impaired energy metabolism resulting in accelerated high energy phosphate depletion during ischemia and delayed recovery of energy equilibrium after cardioplegic arrest observed in hypertrophied hearts could be one of the underlying mechanisms.     

Propofol is cardioprotective in a clinically relevant model of normothermic blood cardioplegic arrest and cardiopulmonary bypass

The general anesthetic propofol has been shown to be cardioprotective. However, its benefits when used in cardioplegia during cardiac surgery have not been demonstrated. In this study, we investigated the effects of propofol on metabolic stress, cardiac function, and injury in a clinically relevant model of normothermic cardioplegic arrest and cardiopulmonary bypass. Twenty anesthetized pigs, randomized to propofol treatment (n = 8) and control (n = 12) groups, were surgically prepared for cardiopulmonary bypass (CPB) and cardioplegic arrest. Doses of warm blood cardioplegia were delivered at 15-min intervals during a 60-min aortic cross-clamped period. Propofol was continuously infused for the duration of CPB and was therefore present in blood cardioplegia. Myocardial biopsies were collected before, at the end of cardioplegic arrest, and 20 mins after the release of the aortic cross-clamp. Hemodynamic parameters were monitored and blood samples collected for cardiac troponin I measurements. Propofol infusion during CPB and before ischemia did not alter cardiac function or myocardial metabolism. Propofol treatment attenuated the changes in myocardial tissue levels of adenine nucleotides, lactate, and amino acids during ischemia and reduced cardiac troponin I release on reperfusion. Propofol treatment reduced measurable hemodynamic dysfunction after cardioplegic arrest when compared to untreated controls. In conclusion, propofol protects the heart from ischemia-reperfusion injury in a clinically relevant experimental model. Propofol may therefore be a useful adjunct to cardioplegic solutions as well as being an appropriate anesthetic for cardiac surgery.     

Improvement of preservation with cardioplegia induced by heat stress is mediated by calcitonin gene-related peptide

Previous studies have shown improvement of preservation with cardioplegia by calcitonin gene-related peptide (CGRP)-induced preconditioning. Therefore we examined the hypothesis that endogenous CGRP may be involved in the protection of heat stress against myocardial damages after prolonged cardioplegic arrest in isolated rat heart. Reperfusion after 4 h of hypothermic ischemia caused a decline of cardiac function and an increase of creatine kinase (CK) release. Heat stress induced by pretreatment with whole body hyperthermia (rectal 42 degrees C) for 15 min produced a significant increase in the plasma content of CGRP, an improvement of cardiac function and a decrease in the release of CK. However, after pretreatment with capsaicin (50 mg/kg, s.c.) to deplete CGRP in cardiac sensory nerves, the plasma concentration of CGRP was no longer increased and the cardioprotection afforded by heat stress was abolished. These findings suggest that improvement of preservation with cardioplegia by heat stress may be mediated by endogenous CGRP in the rat.     

Improved postischemic ventricular functional recovery by amphetamine is linked with its ability to induce heat shock

Heat shock has been shown to increase the cellular tolerances to ischemic injury. In this study, we examined the effects of heat shock induced by amphetamine on postischemic myocardial functional recovery in a setting of coronary revascularization for acute myocardial infarction. Intramuscular injection of amphetamine (3 mg/kg, i.m.) to pigs increased the body temperature to 42.5°C within 1 h, and maintained this temperature for an additional 2 h. Fourty h after the amphetamine injection, the pigs were placed on by cardiopulmonary bypass and then isolated,in situ heart preparations were subjected to 1 h of global hypothermic cardioplegic arrest and 1 h of normothermic reperfusion. Postischemic myocardial performance was monitored by measuring left ventricular (LV) pressure, its dp/dt, myocardial segmental shortening (%SS), and coronary blood flow. Cellular injury was examined by measuring creatine kinase (CK) release. Biochemical measurements included quantification of plasma catecholamines and study of the induction of heat shock gene expression and antioxidative enzymes in the heart tissue. The results of this study indicated significantly greater recovery of LV contractile functions by amphetamine as demonstrated by improved recovery of LVDP (61% vs 52%), dp/dt_max (52% vs 44%), and segmental shortening (46.2% vs 10%). Myocardial CK release was significantly reduced in the amphetamine group. Furthermore, amphetamine pretreatment was associated with the induction of heat shock protein (HSP) 27 mRNA and stimulated Cu/Zn-superoxide dismutase and catalase levels, suggesting that amphetamine mediated improved postischemic ventricular recovery might be linked with its ability to induce heat shock and stimulate antioxidant enzymes.     

Protective effects of a modified apelin-12 and dinitrosyl iron complexes in experimental cardioplegic ischemia and reperfusion

The maintenance of nitric oxide (NO) bioavailability has been recognized as an important component of myocardial protection during cardiac surgery. This study was designed to evaluate the efficacy of using two NO-donating compounds in cardioplegia and reperfusion: (i) a modified peptide apelin-12 (MA12) that activates endothelial NO synthase (eNOS) and (ii) dinitrosyl iron complexes with reduced glutathione (DNIC-GS), a natural NO vehicle. Isolated perfused working rat hearts were subjected to normothermic global ischemia and reperfusion. St. Thomas’ Hospital cardioplegic solution (STH) containing 140 μM MA12 or 100 μM DNIC-GS was used. In separate series, 140 μM MA12 or 100 μM DNIC-GS was administered at early reperfusion. Metabolic state of the hearts was evaluated by myocardial content of high-energy phosphates and lactate. Lactate dehydrogenase (LDH) activity in myocardial effluent was used as an index of cell membrane damage. Cardioplegia with MA12 or DNIC-GS improved recovery of coronary flow and cardiac function, and reduced LDH leakage in perfusate compared with STH without additives. Cardioplegic arrest with MA12 significantly enhanced preservation of high-energy phosphates and decreased accumulation of lactate in reperfused hearts. The overall protective effect of cardioplegia with MA12 was significantly greater than with DNIC-GS. The administration of MA12 or DNIC-GS at early reperfusion also increased metabolic and functional recovery of reperfused hearts. In this case, recovery of cardiac contractile and pump function indices was significantly higher if reperfusion was performed with DNIC-GS. The results show that MA12 and DNIC-GS are promising adjunct agents for protection of the heart during cardioplegic arrest and reperfusion.     

Creatine Kinase-Overexpression Improves Myocardial Energetics,Contractile Dysfunction and Survival in Murine Doxorubicin Cardiotoxicity

Doxorubicin (DOX) is a commonly used life-saving antineoplastic agent that also causes dose-dependent cardiotoxicity. Because ATP is absolutely required to sustain normal cardiac contractile function and because impaired ATP synthesis through creatine kinase (CK), the primary myocardial energy reserve reaction, may contribute to contractile dysfunction in heart failure, we hypothesized that impaired CK energy metabolism contributes to DOX-induced cardiotoxicity. We therefore overexpressed the myofibrillar isoform of CK (CK-M) in the heart and determined the energetic, contractile and survival effects of CK-M following weekly DOX (5mg/kg) administration using in vivo ³¹P MRS and ¹H MRI. In control animals, in vivo cardiac energetics were reduced at 7 weeks of DOX protocol and this was followed by a mild but significant reduction in left ventricular ejection fraction (EF) at 8 weeks of DOX, as compared to baseline. At baseline, CK-M overexpression (CK-M-OE) increased rates of ATP synthesis through cardiac CK (CK flux) but did not affect contractile function. Following DOX however, CK-M-OE hearts had better preservation of creatine phosphate and higher CK flux and higher EF as compared to control DOX hearts. Survival after DOX administration was significantly better in CK-M-OE than in control animals (p<0.02). Thus CK-M-OE attenuates the early decline in myocardial high-energy phosphates and contractile function caused by chronic DOX administration and increases survival. These findings suggest that CK impairment plays an energetic and functional role in this DOX-cardiotoxicity model and suggests that metabolic strategies, particularly those targeting CK, offer an appealing new strategy for limiting DOX-associated cardiotoxicity.     

Nicotinamide riboside kinase-2 alleviates ischemia-induced heart failure through P38 signaling

Nicotinamide riboside kinase-2 (NRK-2), a muscle-specific β1 integrin binding protein, predominantly expresses in skeletal muscle with a trace amount expressed in healthy cardiac tissue. NRK-2 expression dramatically increases in mouse and human ischemic heart however, the specific role of NRK-2 in the pathophysiology of ischemic cardiac diseases is unknown.We employed NRK2 knockout (KO) mice to identify the role of NRK-2 in ischemia-induced cardiac remodeling and dysfunction. Following myocardial infarction (MI), or sham surgeries, serial echocardiography was performed in the KO and littermate control mice. Cardiac contractile function rapidly declined and left ventricular interior dimension (LVID) was significantly increased in the ischemic KO vs. control mice at 2 weeks post-MI. An increase in mortality was observed in the KO vs. control group. The KO hearts displayed increased cardiac hypertrophy and heart failure reflected by morphometric analysis. Consistently, histological assessment revealed an extensive and thin scar and dilated LV chamber accompanied with elevated fibrosis in the KOs post-MI. Mechanistically, we observed that loss of NRK-2 enhanced p38α activation following ischemic injury. Consistently, ex vivo studies demonstrated that the gain of NRK-2 function suppresses the p38α as well as fibroblast activation (α-SMA expression) upon TGF-β stimulation, and limits cardiomyocytes death upon hypoxia/re‑oxygenation.Collectively our findings show, for the first time, that NRK-2 plays a critical role in heart failure progression following ischemic injury. NRK-2 deficiency promotes post-MI scar expansion, rapid LV chamber dilatation, cardiac dysfunction and fibrosis possibly due to increased p38α activation.     

Disorders of cardiac contractile function in ischemic shock; the protective effect of antioxidants and liposomes made from egg phospholipids

Experiments were made on Wistar rats with 6h tourniqueting of the hind limbs to study animal survival rate, myocardial contractile function and protective action of antioxidants and egg phospholipid liposomes during ischemic shock. It has been shown that reperfusion of the limbs leads to a high animal lethality, make lower myocardial contractile function and coronary flow of the hearts isolated from rats following a 6h reperfusion of the limbs. Well-known antioxidant butylated hydroxytoluene and a new antioxidant tetramethylpiperidine derivative bring animal lethality down and improve coronary flow and contractile function of the isolated heart. Phospholipid liposomes increase survival rate moderately but have no any effect on the heart contractile function. It has been deduced that lipid peroxidation takes part in the disturbance of heart contractile function and genesis of the death within ischemic shock.     

Protective effect of peptide semax (ACTH(4-7)Pro-Gly-Pro) on the rat heart rate after myocardial infarction

Semax, a member of ACTH-derived peptides family, was used in treatment of ischemic stroke in patients. It decreased neurological deficiency and reduced NO hyperproduction in the rat brain caused by acute cerebral hypoperfusion. We suggest that semax is also capable of protecting the rat heart from ischemic damage 28 days after myocardial infarction (MI) induced by left descendent coronary artery occlusion. Semax (150 microg/kg) was given i. p. in the operating day twice: 15 min and 2 hours after coronary occlusion, and once a day for the following 6 days. In 28 days after infarction, the MI group developed cardiac hypertrophy, cell growth was caused mainly by the increase of contractile filaments not supported by the appropriate mitochondrial growth that indicated an impaired energy supply of the cells. Moreover, cardiac hypertrophy was accompanied by decreased mean arterial blood pressure and cardiac contractile function and increased left ventricular end-diastolic pressure. Pharmacological change of cardiac afterload revealed that, in 28 days after MI, the rat heart was not able to change its contractile performance in response to either increase or decrease of systemic blood pressure, and as a result could not maintain its diastolic pressure. All these changes obviously reflect development of heart failure. Semax did not affect cardiac work but partially prevented end-diastolic pressure growth in left ventricle as well as ameliorated cardiomyocyte hypertrophy and disproportionate growth of contractile and mitochondrial apparatus, thus exerting beneficial effect on the left ventricular remodeling and heart failure development late after myocardial infarction.     

p38 MAPK-dependent small HSP27 and αB-crystallin phosphorylation in regulation of myocardial function following cardioplegic arrest

We previously demonstrated that myocardial p38 mitogen-activated protein kinase (MAPK) and heat shock protein 27 (HSP27) are phosphorylated following cardioplegic arrest in patients undergoing cardiac surgery and correlate with reduced cardiac function. The following studies were performed to determine whether inhibition of p38 MAPK and/or overexpression of nonphosphorylatable HSP27 improves cardiac function following cardioplegic arrest. Langendorff-perfused isolated rat hearts were subjected to 2 h of intermittent cold cardioplegia followed by 30 min of reperfusion. Hearts were treated with (CP+SB) or without (CP) the p38 MAPK inhibitor SB-203580 (5 μM) supplied in the cardioplegia. Sham-treated hearts served as controls. In separate experiments, isolated rat ventricular myocytes infected with either green fluorescent protein (GFP) or a nonphosphorylatable HSP27 mutant (3A-HSP27) were subjected to 3 h of cold hypoxic cardioplegia and simulated reperfusion (CP) followed by video microscopy and length change measurements. Baseline parameters of cardiac function were similar between groups [left ventricular developed pressure (LVDP), 119 ± 4.9 mmHg; positive and negative first derivatives of LV pressure (± dP/dt), 3,139 ± 245 and 2, 314 ± 110 mmHg/s]. CP resulted in reduced cardiac function (LVDP, 72.2 ± 5.8 mmHg; ± dP/dt, 2,076 ± 231 and -1,317 ± 156 mmHg/s) compared with baseline. Treatment with 5 μM SB-203580 significantly improved CP-induced cardiac function (LVDP, 101.9 ± 0 mmHg; ± dP/dt, 2,836 ± 163 and -2,108 ± 120 mmHg/s; P = 0.03, 0.01, and 0.04, CP+SB vs. CP). Inhibition of p38 MAPK significantly lowered CP-induced p38 MAPK, HSP27, and αB-crystallin (cryAB) phosphorylation. In vitro CP decreased myocyte length changes from 10.3 ± 1.5% (GFP) to 5.7 ± 0.8% (GFP+CP). Infection with 3A-HSP27 completely rescued CP-induced decreased myocyte contraction (11.1 ± 1.0%). However, infection with 3A-HSP27 did not block the endogenous HSP27 response. We conclude that inhibition of p38 MAPK and subsequent HSP27 and cryAB phosphorylation and/or overexpression of nonphosphorylatable HSP27 significantly improves cardiac performance following cardioplegic arrest. Modulation of HSP27 phosphorylation may improve myocardial stunning following cardiac surgery.     

Hypothermic protection of the ischemic heart via alterations in apoptotic pathways as assessed by gene array analysis.

Hypothermia improves resistance to ischemia in the cardioplegia-arrested heart. This adaptive process produces changes in specific signaling pathways for mitochondrial proteins and heat-shock response. To further test for hypothermic modulation of other signaling pathways such as apoptosis, we used various molecular techniques, including cDNA arrays. Isolated rabbit hearts were perfused and exposed to ischemic cardioplegic arrest for 2 h at 34 degrees C [ischemic group (I); n = 13] or at 30 degrees C before and during ischemia [hypothermic group (H); n = 12]. Developed pressure, the maximum first derivative of left ventricular pressure, oxygen consumption, and pressure-rate product (P < 0.05) recovery were superior in H compared with in I during reperfusion. mRNA expression for the mitochondrial proteins, adenine translocase and the beta-subunit of F1-ATPase, was preserved by hypothermia. cDNA arrays revealed that ischemia altered expression of 13 genes. Hypothermia modified this response to ischemia for eight genes, six related to apoptosis. A marked, near fivefold increase in transformation-related protein 53 in I was virtually abrogated in H. Hypothermia also increased expression for the anti-apoptotic Bcl-2 homologue Bcl-x relative to I but decreased expression for the proapoptotic Bcl-2 homologue bak. These data imply that hypothermia modifies signaling pathways for apoptosis and suggest possible mechanisms for hypothermia-induced myocardial protection.     

木犀草素改善低温下离体大鼠心脏保存效果的研究

目的:研究木犀草素是否能改善心脏停搏保存液(UW液)对离体大鼠心脏的低温保存效果。方法:将40只成年SD大鼠随机分成4组(n=10):对照组(UW组)、7.5μmol/L木犀草素小剂量组,15μmol/L木犀草素中剂量组及30μmol/L木犀草素大剂量组。利用Langendorff离体心脏灌流法,观察心脏在4℃含或不含木犀草素的UW液中保存12 h复灌60 min后心脏功能及超微结构变化,比较心脏冠脉流量(CF)、心肌含水量及冠脉流出液中磷酸肌酸激酶(CK)的释放量。结果:与对照组比较,添加木犀草素后,复灌期心脏的收缩功能(LVPSP,+dp/dtmax)与心脏舒张功能(-dp/dtmax)、冠脉流量在多个复灌时间点均优于对照组,心率在复灌60 min时也显著优于对照组;复灌过程中磷酸肌酸激酶的漏出量及低温保存后心脏超微结构的损伤也均明显低于对照组;随灌注时间延长木犀草素组心脏结构和功能的改善有剂量依赖性趋势;木犀草素对心肌含水量没有影响。结论:木犀草素能显著改善UW液对离体大鼠心脏的低温保存效果,对心脏有明显的保护作用,以30μmol/L的木犀草素大剂量组作用最显著。     

Temperature Threshold and Preservation of Signaling for Mitochondrial Membrane Proteins during Ischemia in Rabbit Heart

Temperature modulates both myocardial energy requirements and production. We have previously demonstrated that myocardial protection induced by hypothermic adaptation preserves expression of genes regulating heat shock protein and the nuclear-encoded mitochondrial proteins, the adenine nucleotide translocator isoform 1 (ANT₁), and the β subunit of F₁-ATPase (βF₁-ATPase). This preservation is associated with a reduction in ATP depletion similar to that noted in cardioplegic arrested hearts preserved at a critical temperature (30°C) or below. We tested the hypothesis that expression of these genes may also be subject to this temperature threshold phenomenon. Isolated perfused rabbit hearts were subjected to ischemic cardioplegic arrest at 4, 30, or 34°C for 120 min. Cardiac function indices and steady-state mRNA levels for ANT₁, βF₁-ATPase, and HSP70-1 were measured prior to ischemia (B) and after 45 min of reperfusion. Cardiac function was significantly depressed in the 34°C group. Ischemia at 34°C reduced steady-state mRNA levels for ANT₁and βF₁-ATPase from B, but these levels were similarly preserved at 4 and 30°C. HSP70-1 levels were mildly elevated (fourfold) above B to similar levels at all three temperatures. These results indicate that mRNA expression for ANT₁and βF₁-ATPase is specifically preserved in a pattern consistent with the temperature threshold phenomenon. HSP70-1 expression is not influenced by ischemic temperature. Preservation of gene expression for these mitochondrial proteins implies that signaling for mitochondrial biogenesis or resynthesis is maintained after ischemic insult.     

二氮嗪在长时程心脏低温保存中的作用

延长心脏的体外有效保存时间对临床心脏移植具有重要意义。本文旨在研究线粒体ATP敏感性钾通道开放剂二氮嗪(diazoxide,DE)在离体大鼠心脏长时程低温保存中的作用。SD大鼠随机分成5组,包括对照组(单纯Celsior保存液),DE组(Celsior液中含15、30或45μmol／L的DE)和DE 5-HD组[Celsior液中含30μmol／L的DE和100μmol／L的5-羟基葵酸盐(5-hydroxydecanoate,5-HD)]。利用Langendorff离体鼠心灌注法,观察心脏在4℃条件下保存10h后,复灌期血流动力学恢复、冠脉流出液中心肌酶漏出量及心肌水含量变化,并做心肌超微结构检查。结果显示：与对照组比较,DE处理后,复灌期的左心室舒张末期压力明显降低,心率、左心室发展压、左心室压力变化率、冠脉流出量等的恢复率在多个复灌时间点上优于对照组,且能显著减少复灌过程中心肌酶(乳酸脱氢酶、磷酸肌酸激酶及谷草转氨酶)的漏出量,降低心肌水含量;其中30和45μmol／LDE组的保护作用优于15μmol／LDE组;电镜结果显示DE对长时程低温保存心脏的超微结构有较好的保护作用。DE的上述作用可被线粒体ATP敏感性钾通道的特异性阻断剂5-HD所取消。以上结果提示：DE可通过激活线粒体ATP敏感性钾通道显著改善离体大鼠心脏长时程低温保存效果。