Involvement of sodium in the protective effect of 5-(N,N-dimethyl)-amiloride on ischemia-reperfusion injury in isolated rat ventricular wall

During reperfusion in the isolated right ventricular wall of the rat after 60 min of ischemia, developed tension and resting tension were 35 +/- 4 and 221 +/- 12%, respectively, of preischemic values. Including 35 microM ouabain in the perfusate before and after ischemia resulted in more severe cardiac dysfunction during reperfusion than in drug-untreated hearts. Introduction of the Na(+)-H+ exchange inhibitor, 5-(N,N-dimethyl)-amiloride (DMA), could effectively protect the right ventricular wall against ischemia-reperfusion dysfunction in the presence or absence of ouabain. The ion content in the right ventricular wall was measured with atomic absorbance spectrophotometry. Before ischemia, Na+,Ca++ and K+ content were 53.4 +/- 6.4, 2.70 +/- 0.22 and 262 +/- 7.7 mumol/g of dry weight tissue, respectively. After 60 min of ischemia and 6 min of reperfusion, Na+,Ca++ and K+ content were 73.4 +/- 7.2, 3.79 +/- 0.31 and 180 +/- 15 mumol/g of dry weight tissue, respectively (P less than .05). Introduction of 20 microM DMA normalized ion content in the muscles which was consistent with the contractile function recovery during reperfusion. The data suggest that a rise in intracellular Na+ in the early stage of reperfusion represents a crucial or primary step for the development of cardiac contractile dysfunction. DMA, which protects against severe reperfusion-induced cardiac contractile dysfunction, appears to act via a normalization of tissue sodium levels. This action is consistent with its proposed role as a blocker of transsarcolemmal Na(+)-H+ exchange.     

Oxygen-derived free radicals and postischemic myocardial reperfusion: therapeutic implications

Oxygen-derived free radicals have been implicated in the pathogenesis of various disease states, including myocardial ischemia and reperfusion. In this article, we review 1) the evidence linking free radical production and myocardial injury during myocardial ischemia and reperfusion and 2) results of studies of the effects of the pharmacological therapies available potentially to prevent free radical-mediated injury. Free radicals can be produced during ischemia and reperfusion by several different biochemical pathways. Of these, the xanthine oxidase reaction and the output of free radicals by neutrophils that have accumulated in damaged tissue have been studied extensively. When produced, free radicals can potentially damage myocytes or endothelial cells through peroxidation of membrane lipids or damage to proteins or nucleic acids. Using electron spin resonance spectroscopy, several studies have shown a 'burst' of oxygen free radicals immediately after reperfusion. Moreover, exogenous generation of intravascular free radicals has been shown to produce marked vascular and myocyte damage, as well as contractile dysfunction. 'Anti-free radical' interventions, such as xanthine oxidase inhibitors and free radical scavengers have been reported to prevent contractile dysfunction and reperfusion-induced arrhythmias after an episode of reversible ischemic injury. However, after more severe episodes of ischemia, such interventions have had conflicting effects on myocardial infarct size. 'Anti-free radical' interventions could be of potential use in situations where reversible ischemic injury occurs. In situations where reperfusion is achieved after irreversible ischemic injury has occurred, the potential beneficial effect of these treatments on infarct size is more doubtful.     

The beneficial effect of 2′-deoxycoformycin in renal ischemia-reperfusion is mediated both by preservation of tissue ATP and inhibition of lipid peroxidation

Renal ischemia injures the renal tubular cell by disrupting the vital cellular metabolic machinery. Further cell damage is caused when the blood flow is restored by oxygen free radicals that are generated from xanthine oxidase. Oxygen radicals cause lipid peroxidation of cell and organelle membranes, disrupting the structural integrity and capacity for cell transport and energy metabolism. In the present study, the possible therapeutic usefulness of the adenosine deaminase inhibitor, 2'-deoxycoformycin (DCF), during renal ischemia and reperfusion injury was investigated. The effects of DCF on renal malondialdehyde (MDA) and ATP levels were studied after 45 min ischemia and 15 min subsequent reperfusion in rat kidneys. MDA levels remained unchanged during ischemia, but increased after the subsequent reperfusion. DCF pretreatment (2.0 mg/kg i.m.) decreased MDA and increased ATP levels during the ischemia-reperfusion period. DCF exerts a dual protective action by facilitating purine salvage for ATP synthesis and inhibiting oxygen radical-induced lipid peroxidation. These results suggest that DCF therapy could be beneficial in the treatment of ischemia-reperfusion renal injuries.     

Na~+-H~+交换抑制剂在心肌缺血与再灌注损伤中的研究进展

抑制肌膜Na+ H+ 交换能减少心肌缺血与再灌注损伤。动物实验显示 ,Na+ H+ 交换抑制剂能减少心肌缺血与再灌注时的严重心律失常发生率 ,延缓心功能不全的发生 ,限制组织坏死。临床试验显示 ,Na+ H+ 交换抑制剂可减少心肌梗死后再灌注损伤 ,改善左室功能不全的恢复     

Effect of the phospholipase A2 inhibitors quinacrine and 7,7-dimethyleicosadienoic acid in isolated globally ischemic rat hearts.

Phospholipase A2 (PLA2) activity results in the formation of lysophospholipids and free fatty acids which may contribute to ischemic myocardial dysfunction. We evaluated the cardioprotective activity of two putative PLA2 inhibitors, quinacrine and 7,7-dimethyleicosadienoic acid (DEDA), in isolated globally ischemic rat hearts. Pretreatment with 1, 5 and 50 microM quinacrine before ischemia did not alter coronary flow but did cause significant cardiodepression. Twenty five minutes of global ischemia and 30 min of reperfusion caused severe myocardial dysfunction and lactate dehydrogenase release. Quinacrine significantly improved reperfusion contractile function and reduced lactate dehydrogenase release, indicative of cardioprotection. In contrast, 30 to 100 microM DEDA produced neither preischemic cardiodepression nor cardioprotective activity. PLA2 inhibition was inferred from measurements of the prostacyclin metabolite, 6-keto-prostaglandin F1 alpha in the coronary effluent and myocardial palmitoyl-lysophosphatidylcholine. Quinacrine and DEDA reduced both 6-keto-prostaglandin F1 alpha and palmitoyl-lysophosphatidylcholine by similar degrees. These results suggest that the cardioprotective activity of quinacrine is independent of PLA2 inhibition. A possible role of calcium inhibition was investigated in rat aortic smooth muscle strips. Norepinephrine-, KCl- and BAY K8644-induced contractions were antagonized in the presence of 5 and 50 microM quinacrine, but were unaffected by 30 to 60 microM DEDA. The ability of quinacrine to inhibit calcium was investigated further in cardiac ventricular myocytes. Measurement of mean whole cell calcium currents showed that quinacrine (5 microM) could inhibit this current up to 70%. Thus, these results suggest that quinacrine-induced cardioprotection may not be due to PLA2 inhibition, but may be related to calcium entry blocking activity.     

Lactate activates ATP-sensitive potassium channels in guinea pig ventricular myocytes.

The functional significance of cardiac ATP-sensitive potassium channels remains controversial because of the discrepancy between the low levels of ATP at which activation of the channels occurs and the much higher levels of ATP maintained during myocardial ischemia. We studied the effects of (+)-lactate, which accumulates in large quantity as a result of increased glycolysis during ischemia, on ATP-sensitive potassium channels in adult guinea pig ventricular myocytes using the whole-cell patch-clamp technique. Lactate at 20-40 mM in the internal solution activated ATP-sensitive potassium channels and shortened action potential duration. Activation of the channels occurred even in the presence of 2-5 mM ATP in the internal solution and was dependent on intracellular free magnesium levels. Our results suggest that intracellular lactate may play a significant role in activating cardiac ATP-sensitive potassium channels and shortening action potential duration even at ATP levels similar to those resulting from moderate to severe myocardial ischemia.     

Activation of ATP-sensitive outward K+ current by nicorandil (2-nicotinamidoethyl nitrate) in isolated ventricular myocytes

The vasodilatory agent, nicorandil (2-nicotinamidoethyl nitrate) activates an outward K+ current in cardiac and vascular smooth muscle. This current was studied with the patch clamp technique using isolated guinea pig and rabbit ventricular myocytes. Nicorandil (10(-5) and 10(-4) M) shortened the action potential duration without any significant change in the resting membrane potential. Under voltage clamp, nicorandil increased the time-independent outward current at potentials positive to -80 mV, and decreased the inward current at potentials negative to -90 mV. The drug did not affect Ca++ current activated upon depolarization from the holding potential of -30 mV, or did it influence delayed outward K+ current on repolarization. In rabbit myocytes, nicorandil did not increase the Ca++-sensitive and -insensitive transient outward K+ currents. When the ATP concentration of the pipette solution was reduced from 5 to 2 to 3 mM, nicorandil produced a large increase in outward current, which decreased slightly with time. The increased outward current was antagonized by raising the intracellular ATP concentration. Nicorandil increased the probability of opening of the ATP-sensitive single channel current without affecting its unitary amplitude. These results indicate that nicorandil activates the ATP-sensitive K+ current, which is responsible for shortening of the action potential duration.     

Glycolytic inhibition and calcium overload as consequences of exogenously generated free radicals in rabbit hearts.

Free radicals have been implicated in the pathogenesis of reperfusion injury, but it is unclear how they exert their deleterious effects on cellular metabolism. Several lines of indirect evidence suggest that free radicals elevate intracellular Ca2+ concentration ([Ca2+]i) and inhibit glycolysis as part of their mechanism of injury. We tested these ideas directly in hearts subjected to hydroxyl radicals produced by the Fenton and Haber-Weiss reactions. Nuclear magnetic resonance spectra were obtained from Langendorff-perfused rabbit hearts before, during, and after 4 min of perfusion with H2O2 (0.75 mM) and Fe(3+)-chelate (0.1 mM). Isovolumic left ventricular pressure exhibited progressive functional deterioration and contracture after exposure to H2O2 + Fe3+. Phosphorus nuclear magnetic resonance (NMR) spectra revealed partial ATP depletion and sugar phosphate accumulation indicative of glycolytic inhibition. To measure [Ca2+]i, fluorine NMR spectra were acquired in a separate group of hearts loaded with the Ca2+ indicator 5F-BAPTA [5,5'-difluoro derivative of 1,2-bis-(o-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid]. Mean time-averaged [Ca2+]i increased from 347 +/- 14 nM in control to 1,026 +/- 295 nM 4 min after free radical generation (means +/- SEM, n = 7), and remained elevated thereafter. We conclude that free radicals induce clear-cut, specific derangements of cellular metabolism in the form of glycolytic inhibition and calcium overload. The observed increase in [Ca2+]i suggests that the deleterious effects of free radicals are at least partially mediated by secondary changes in cellular calcium homeostasis.     

二维超声心动图评价局部心肌缺血的新指标

心肌缺血引起局部左室收缩功能减低常伴有心室内径增大和局部室壁变薄。收缩末期半径与局部室壁厚度比值（Ｒ／Ｔｈ）是二维超声心动图（２ＤＥ）评价局部左室收缩功能的一种新指标，并不受心脏位置移动的影响。１２只麻醉犬用自制微米缩窄器造成暂时性不同程度心肌缺血。以２ＤＥ短轴切面等分１２节段分别计测收缩末期Ｒ／Ｔｈ和半轴缩短率。在心肌缺血区，收缩末期Ｒ／Ｔｈ缺血前平均１．５０±０．７０％，缺血时增加至２．２０＋０．８％，再灌注后逐渐恢复正常；局部半径缩短率从正常时平均３８．１±８．２％减低至１．７±７．９％，再灌注后逐渐恢复到基础状态。实验发现收缩末期Ｒ／Ｔｈ和室壁增厚率及半轴缩短率各自相关良好（Ｒ＝－０．８８和０．７６）。结论：测定收缩末期Ｒ／Ｔｈ为定量局部左室收缩功能提供一种超声心动图指标，与现在的２ＤＥ方法比较，具有独特的判断标准并且不受心脏移动的影响。     

Mechanism of hypoxic K loss in rabbit ventricle.

Although a critical factor causing lethal ischemic ventricular arrhythmias, net cellular K loss during myocardial ischemia and hypoxia is poorly understood. We investigated whether selective activation of ATP-sensitive K (KATP) channels causes net cellular K loss by examining the effects of the KATP channel agonist cromakalim on unidirectional K efflux, total tissue K content, and action potential duration (APD) in isolated arterially perfused rabbit interventricular septa. Despite increasing unidirectional K efflux and shortening APD to a comparable degree as hypoxia, cromakalim failed to induce net tissue K loss, ruling out activation of KATP channels as the primary cause of hypoxic K loss. Next, we evaluated a novel hypothesis about the mechanism of hypoxic K loss, namely that net K loss is a passive reflection of intracellular Na gain during hypoxia or ischemia. When the major pathways promoting Na influx were inhibited, net K loss during hypoxia was almost completely eliminated. These findings show that altered Na fluxes are the primary cause of net K loss during hypoxia, and presumably also in ischemia. Given its previously defined role during hypoxia and ischemia in promoting intracellular Ca overload and reperfusion injury, this newly defined role of intracellular Na accumulation as a primary cause of cellular K loss identifies it as a central pathogenetic factor in these settings.     

Tedisamil blocks the transient and delayed rectifier K+ currents in mammalian cardiac and glial cells

The potassium currents in rat and guinea pig ventricular myocytes and mouse astrocytes were studied using tedisamil, a novel antiarrhythmic agent. A 1 to 20 microM dosage of tedisamil caused marked prolongation of the action potential in isolated rat ventricular myocytes, mimicking its reported effects on multicellular rat heart preparations. Under voltage clamp conditions, tedisamil caused a dose-dependent increase in the speed of inactivation of the transient outward K+ current (Ito), the predominant outward current in rat ventricular myocytes. In cardiac myocytes, the tedisamil block was neither use- nor voltage-dependent. The slow reversibility of drug action when applied from the outside, and its effectiveness when applied intracellularly, suggested an internal site of drug action. In guinea pig ventricular myocytes, tedisamil blocked the slowly developing time-dependent delayed rectifier K+ current (IK) over the same concentration range as that found for Ito in the rat myocytes. Tedisamil reduced this current without changing the characteristics of its slow (tau approximately 1 sec) activation. The effects of tedisamil on Ito and IK were independent of the phosphorylation state of the channel, as assessed by the equal effectiveness of the drug in the presence or absence of isoproterenol. Tedisamil also blocked the transient K+ current and the delayed rectifier current (IK) in mouse astrocytes over the same concentration range as that found in the cardiac myocytes and by a process that accelerated (transient K+ current) or mimicked (IK) inactivation. At concentrations of up to 50 microM, tedisamil had little effect on the time-dependent inward rectifier K+ current, or inward calcium current in rat or guinea pig ventricular myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular calcium and ventricular function. Effects of nisoldipine on global ischemia in the isovolumic, coronary-perfused heart.

Ischemia-induced ventricular dysfunction has been shown to be associated with increased diastolic and systolic intracellular concentrations of free, ionized calcium ([Ca2+]i). The present study was designed to determine the effects of the Ca2+ antagonist nisoldipine on the relationship between [Ca2+]i and left ventricular contraction and relaxation during ischemia and reperfusion on a beat-to-beat basis. Nine isovolumic coronary-perfused ferret hearts were made globally ischemic for 3 min and reperfused for 10 min. Ischemia and reperfusion were repeated during perfusion with a buffer containing 10(-8) M nisoldipine. From left ventricular developed pressure, time to peak pressure and time to 50% pressure decline were obtained. [Ca2+]i was determined with the bioluminescent protein aequorin. Global ischemia caused a rapid decline in contractile function and a significant increase in diastolic [Ca2+]i, from 0.35 to 0.81 microM, and in systolic [Ca2+]i, from 0.61 to 0.96 microM. During reperfusion, [Ca2+]i returned to baseline while ventricular function was still impaired. Relaxation was more affected than systolic contractile function. Nisoldipine significantly reduced the ischemia-induced rise in diastolic [Ca2+]i to 0.62 microM, and in systolic [Ca2+]i to 0.77 microM, and lessened the decrease in contractile function. Nisoldipine significantly accelerated the decline in [Ca2+]i during reperfusion and improved recovery of contractility and relaxation. These effects were associated with a significant diminution in ischemic lactate production. Taken together, our results provide direct quantitative evidence on a beat-to-beat basis that the calcium antagonist nisoldipine can ameliorate ischemia-induced abnormalities in [Ca2+]i handling, an effect that was associated with improved myocardial function during early reperfusion.     

Effects of lysophosphatidylcholine on electrophysiological properties and excitation-contraction coupling in isolated guinea pig ventricular myocytes.

Lysophosphoglyceride accumulation in ischemic myocardium has been implicated as a cause of arrhythmias. We examined the effects of lysophosphatidylcholine (LPC) in isolated guinea pig ventricular myocytes. In paced myocytes loaded with the Ca2+ indicator Indo-1-AM and studied at room temperature, 20 microM LPC caused an initial positive inotropic effect followed by spontaneous automaticity, a decline in active cell shortening, and progressive diastolic shortening (contracture) leading to cell death. These changes were accompanied by a progressive increase in cytosolic [Ca2+]i. In patch-clamped myocytes dialyzed internally with high EGTA concentrations, LPC caused membrane depolarization, shortening of the action potential duration, and abnormal automaticity as seen in multicellular preparations. Voltage clamp experiments revealed the appearance of a nonselective leak conductance without significant changes in the delayed rectifier K+ current, inward rectifier K+ current, L-type Ca2+ current, and T-type Ca2+ current. Pretreatment with 20 mM caffeine and [Ca2+]o-free solution did not prevent the leak current. In patch clamped myocytes loaded with 0.1 mM Fura-2 salt, the [Ca2+]i transient induced by either voltage clamps or brief caffeine exposure remained normal until the nonselective leak current developed. The Na(+)-Ca2+ exchange current elicited during caffeine-induced [Ca2+]i transients also did not appear to be altered by LPC. Qualitatively similar results were obtained in myocytes studied at 35 degrees C. The membrane detergent saponin (0.005% wt/wt) mimicked all of the effects of LPC. We conclude that under these experimental conditions the effects of LPC are most compatible with a detergent action causing membrane leakiness with resultant depolarization, [Ca2+]i overload, and contracture.     

Xanthine oxidase produces hydrogen peroxide which contributes to reperfusion injury of ischemic, isolated, perfused rat hearts.

Three lines of investigation indicated that hydrogen peroxide (H2O2) from xanthine oxidase (XO) contributes to cardiac dysfunction during reperfusion after ischemia. First, addition of dimethylthiourea (DMTU), a highly permeant O2 metabolite scavenger (but not urea) simultaneously with reperfusion improved recovery of ventricular function as assessed by ventricular developed pressure (DP), contractility (+dP/dt), and relaxation rate (-dP/dt) in isolated Krebs-Henseleit-perfused rat hearts subjected to global normothermic ischemia. Second, hearts from rats fed tungsten or treated with allopurinol had negligible XO activities (less than 0.5 mU/g wet myocardium compared with greater than 6.0 mU/g in control hearts) and increased ventricular function after ischemia and reperfusion. Third, myocardial H2O2-dependent inactivation of catalase occurred after reperfusion following ischemia, but not after ischemia without reperfusion or perfusion without ischemia. In contrast, myocardial catalase did not decrease during reperfusion of ischemic hearts treated with DMTU, tungsten, or allopurinol.     

Shortening deactivation of cardiac muscle: physiological mechanisms and clinical implications.

PHYSIOLOGICAL MECHANISM: A rapid change of length applied during isometric contraction of skeletal or cardiac muscle may result in redeveloped tension less than appropriate for the new length because of "deactivation" of the contractile system. The amount of shortening deactivation is directly related to both the time during the contraction when the length change occurs and to the extent of muscle shortening. If the muscle is permitted to shorten early in the contraction, the redeveloped tension will be appropriate to the new length as predicted from the classic Frank-Starling relationship. However, the same length change, which is imposed later in the contraction, results in a redeveloped tension that is less than predicted. Furthermore, a greater change in length results in less tension being redeveloped than if a smaller length decrement is applied at the same time during the contraction. It has been demonstrated that the reduced tension during active muscle shortening is associated with reduced affinity of troponin C for Ca2+. The free Ca2+ is then picked up by the SR, with less Ca2+ available for tension development until the subsequent contraction. CLINICAL SIGNIFICANCE: Although the clinical significance of shortening deactivation remains speculative, it seems likely that in the intact heart deactivation would affect myocardial O2 consumption. The decreased efficiency with which the heart maintains a given stroke work against a high afterload might be related to the lesser degree of fiber shortening and, therefore, less shortening deactivation. Conversely, it is well-known that the same level of stroke work accomplished by an increase in end-diastolic volume requires much less O2. This may be related, at least in part, to the greater degree of shortening with an accompanying increase in deactivation under the latter conditions. For example, in congestive heart failure where ejection fraction and fiber shortening are minimal, the maintenance of the longer fiber lengths could significantly increase the MVO2. Ford has suggested that the deactivating effect of shortening produced by afterload reduction would limit energy expenditure, therefore, exerting a favorable effect on the failing myocardium. It would also seem that an inotropic agent that increased shortening deactivation might compensate for the increased MVO2 caused by the inotrope and have a favorable effect on cardiac work. From most of the studies we have reviewed, it appears likely that shortening deactivation acts as a physiological "feedback" mechanism that affects afterload and in turn, myocardial oxygen consumption. Pathological situations such as acidosis and ischemia have been associated with reduced myofilament Ca2+ sensitivity or affinity and depressed cardiac contractility. Is it then possible that interventions that increase Ca2+ sensitivity might favorably alter ventricular pressure-volume relations during ejection and improve myocardial function by reducing the magnitude of shortening deactivation? Whatever the mechanism and clinical significance, future investigations will help to define the role of shortening deactivation in modifying ventricular function.     

丙泊酚脑保护作用机制研究进展

脑缺血再灌注、急性颅脑损伤、体外循环等可导致脑组织不同程度的损伤,其机制主要包括氧化应激、能量代谢异常、钙离子超载、炎症细胞和炎症因子的作用、血管舒缩因子失衡、蛋白和酶表达异常等。丙泊酚是目前应用最广泛的静脉麻醉药,具有一定的脑保护作用,可能机制有降低脑代谢、改善脑血管功能、抗自由基、抑制脂质过氧化、抑制细胞内钙超载、抑制炎性细胞因子的表达、抑制细胞凋亡等。     

Mesenteric lymph from rats with thermal injury prolongs the action potential and increases Ca2+ transient in rat ventricular myocytes

Although gut-derived mesenteric lymph from animals with thermal injury appears to lead to myocardial contractile dysfunction, the cellular mechanisms remain unclear. We examined the direct effects of intestinal lymph on excitation-contraction coupling in rat ventricular myocytes. Lymph from rats receiving burn injury (burn lymph), but not from sham-burned rats, rapidly enhanced myocyte contraction and the amplitude of Ca2+ transient; the average percentage of shortening was increased from 5.5 +/- 0.3% to 10.5 +/- 0.9%. 90% and the Ca2+ transients increased by 80% +/- 20%. Burn lymph had no effect on the amplitude of L-type Ca2+ current (ICa) or the inward rectifier K+ current, but the transient outward K+ currents (Ito) were reduced significantly by burn lymph. Inhibition of Ito was not altered by an alpha1-adrenergic receptor (AR) antagonist, prazosin, indicating that the block was not mediated via alpha1-AR signaling pathway. Action potential (AP) duration, measured at 50% and 90% repolarization, was prolonged by burn lymph. Stimulation of myocytes with AP voltage-clamp waveforms derived from prolonged AP induced by burn lymph revealed a 1.7-fold increase in Ca2+ influx via ICa compared with the Ca2+ influx induced by control AP. Blocking of Ito by 4-aminopyridine prolonged AP duration and increased Ca2+ transients, mimicking the effects of burn lymph. Burn lymph did not affect Na+/Ca2+ exchange currents or caffeine-induced SR Ca2+ release. Thus, acute exposure of normal cardiac myocytes to burn lymph increases Ca2+ transients by a prolongation of AP as a result of a reduction of Ito with no intrinsic change in ICa or exchanger. The electrophysiological changes are similar to those that occur during compensated cardiac hypertrophy, suggesting a common mechanistic link between burn lymph- and hypertrophy-induced cardiac dysfunction.     

Demonstration of free radical generation in "stunned" myocardium of intact dogs with the use of the spin trap alpha-phenyl N-tert-butyl nitrone

Recent studies suggest that oxygen free radicals may mediate postischemic myocardial dysfunction ("stunning"), but all the evidence for this hypothesis is indirect. Thus, we used electron paramagnetic resonance (EPR) spectroscopy and the spin trap, alpha-phenyl N-tert-butyl nitrone (PBN), to directly investigate whether free radicals are produced after a 15-min coronary artery occlusion and subsequent reperfusion in 30 open-chest dogs. After intracoronary infusion of PBN, EPR signals characteristic of oxygen- and carbon-centered radical adducts were detected in the venous blood draining from the ischemic/reperfused vascular bed. The myocardial release of PBN adducts began during coronary occlusion but increased dramatically in the first few minutes after reperfusion. After this initial burst, the production of radicals abated but did not cease, persisting up to 3 h after reflow. The EPR spectra (aH beta = 2.67-2.79 G, aN = 14.75-15.00 G) were consistent with the trapping by PBN of secondary oxygen- and carbon-centered radicals, such as alkoxy and alkyl radicals, which could be formed by reactions of primary oxygen radicals with membrane lipids. There was a linear, direct relationship between the magnitude of PBN adduct production and the degree of ischemic flow reduction. Recovery of contractile function (measured as systolic wall thickening) after reperfusion was greater (P less than 0.05) in dogs given PBN than in controls. This study demonstrates that reversible regional myocardial ischemia in the intact animal is associated with prolonged free radical generation, and that the intensity of such generation is related to the severity of ischemia. The results provide direct evidence to support the hypothesis that reactive oxygen metabolites contribute to the persistent contractile dysfunction (myocardial stunning) observed after brief ischemia in vivo.     

Cyclosporine and FK506 differentially regulate the sarcolemmal Na(+)-K(+) pump

Cyclosporine A (CsA) and FK506, important immunosuppressants, have been shown to inhibit the enzymatic equivalent of the Na(+)-K(+) pump (Na(+), K(+)-ATPase) in renal tissue. A similar effect in the heart may contribute to the adverse effects of these agents that include calcification, contractile dysfunction, and altered calcium handling. However, inhibition of the pump has not been demonstrated in cardiac myocytes. We isolated single ventricular myocytes from control rabbits and from rabbits administered CsA or FK506 for 1 week. Na(+)-K(+) pump current (I(p)) was measured using the whole-cell patch-clamp technique. When patch pipettes contained Na(+) in a concentration ([Na](pip)) near physiological intracellular levels mean I(p) of cardiac myocytes from rabbits with serum CsA levels within the therapeutic range was significantly lower than mean I(p) of cardiac myocytes from controls. Treatment had no effect on I(p) measured using a [Na](pip) expected to nearly saturate intracellular binding sites. The CsA-induced inhibition of I(p) was dependent on the K(+) concentration in pipette solutions. Mean I(p) in myocytes from rabbits with serum levels of FK506 within the therapeutic range was similar to mean I(p) in myocytes from controls, whereas FK506 in a dose inducing serum levels severalfold above the therapeutic range caused significant pump inhibition. Using ion-sensitive microelectrodes we showed the intracellular Na(+) activity in papillary muscles isolated from rabbits treated with CsA was significantly higher than in papillary muscles from control rabbits, indicating that CsA causes pump inhibition in intact myocytes with a physiological intracellular milieu.     

Reperfusion injury induces apoptosis in rabbit cardiomyocytes.

The most effective way to limit myocardial ischemic necrosis is reperfusion, but reperfusion itself may result in tissue injury, which has been difficult to separate from ischemic injury. This report identifies elements of apoptosis (programmed cell death) in myocytes as a response to reperfusion but not ischemia. The hallmark of apoptosis, nucleosomal ladders of DNA fragments (approximately 200 base pairs), was detected in ischemic/reperfused rabbit myocardial tissue but not in normal or ischemic-only rabbit hearts. Granulocytopenia did not prevent nucleosomal DNA cleavage. In situ nick end labeling demonstrated DNA fragmentation predominantly in myocytes. The pattern of nuclear chromatin condensation was distinctly different in reperfused than in persistently ischemic tissue by transmission electron microscopy. Apoptosis may be a specific feature of reperfusion injury in cardiac myocytes, leading to late cell death.