Pharmacological preconditioning with diazoxide slows energy metabolism during sustained ischemia

Ischemic preconditioning (PC) is associated with slower destruction of the adenine nucleotide pool ( summation operatorAd) and slower rate of anaerobic glycolysis during ischemic stress. These changes are concordant with the preconditioned state, supporting an essential role of lowered energy demand in the cardioprotective mechanism of PC. Although pharmacological PC induced by the activation of mitochondrial K(ATP) channels also limits infarct size, its effect on energy metabolism during sustained ischemia is unknown. Using metabolite levels found at baseline and after a 15 min test episode of regional ischemia, the effect of a cardioprotective dose of diazoxide on metabolic features associated with PC was tested in barbital-anesthetized, open-chest dogs. Diazoxide (3.5 mg/kg at an intravenous rate of 1 mL/min) infused before a test episode of ischemia had no effect on baseline metabolic indices. However, during ischemic stress, treated hearts exhibited less destruction of ATP, less degradation of the summation operatorAd into nucleosides and bases, as well as less lactate production than control hearts subjected only to ischemic stress. Thus, diazoxide mimics the metabolic alterations observed in PC tissue. This supports the hypothesis that a reduction in energy demand, which is now equated with an increased ATP to ADP ratio in the sarcoplasm, is a critical component of the mechanism of cardioprotection in preconditioned myocardium. It is hypothesized that during PC or diazoxide treatment, the passage of the summation operatorAd into and out of the mitochondria is slowed, limiting the level of ATP available to the mitochondrial ATPase and preserving ATP and the total summation operatorAd. Altered ischemic mitochondrial metabolism plays an important role in establishing and maintaining the preconditioned state.     

Cytochrome P450 omega-hydroxylase inhibition reduces infarct size during reperfusion via the sarcolemmal KATP channel

Inhibition of 20-hydroxyeicosatrienoic acid (20-HETE), by pretreatment with pharmacological inhibitors of cytochrome P450 (CYP) omega-hydroxylase, has been shown to reduce infarct size in canines when administered prior to ischemia. However, it is unknown whether these agents reduce infarct size when administered just prior to reperfusion and if the sarcolemmal and/or mitochondrial K(ATP) channels (sK(ATP) and mK(ATP)) contribute to cardioprotection. Therefore, we determined whether specific CYP inhibitors for epoxygenases and omega-hydroxylases are cardioprotective when given either prior to ischemia or prior to reperfusion and furthermore, if selective inhibition of the sK(ATP) by HMR-1098 or mK(ATP) by 5-hydroxydecanoic acid (5-HD) could abrogate this effect. Male Sprague-Dawley rats underwent 30 minutes of ischemia followed by 2 hours of reperfusion. Groups received either miconazole (MIC, non-selective CYP inhibitor, 3 mg/kg), 17-octadecynoic acid (17-ODYA, CYP omega-hydroxylase inhibitor, 0,3 or 3 mg/kg), N-methylsulfonyl-12, 12-dibromododec-11-enamide (DDMS, CYP omega-hydroxylase inhibitor, 0,4 or 4 mg/kg), N-methanesulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH, CYP epoxygenase inhibitor, 3 mg/kg), or vehicle either 10 minutes prior to ischemia or 5 minutes prior to reperfusion. Rats also received either HMR-1098 (6 mg/kg) or 5-HD (10 mg/kg) 10 minutes prior to reperfusion, with subsets of rats also receiving either MIC or 17-ODYA 5 minutes prior to reperfusion. DDMS and 17-ODYA dose dependently reduced infarct size. Rats treated with MIC, 17-ODYA and DDMS, but not MS-PPOH, produced comparable reductions in infarct size when administered prior to ischemia or reperfusion compared to vehicle. HMR-1098, but not 5-HD, also blocked the infarct size reduction afforded by MIC and 17-ODYA. These data suggest a novel cardioprotective pathway involving CYP omega-hydroxylase inhibition and subsequent activation of the sK(ATP) channel during reperfusion.     

Molecular oxygen: friend and foe. The role of the oxygen free radical system in the calcium paradox, the oxygen paradox and ischemia/reperfusion injury

We strongly support the original intriguing hypothesis of Hearse et al. that the oxygen paradox and the calcium paradox are facets of the same problem. We would propose that the major similarity is a final common pathway leading to intracellular calcium overload and the sequelae of the resultant increase in intracellular calcium. In addition, we would propose that the oxygen paradox and ischemic/reperfusion injury are also facets of the same problem with the major similarity being the reintroduction of molecular oxygen to a previously hypoxic myocardium. Finally, we would suggest that the common pathway leading to intracellular calcium overload in the oxygen paradox and ischemic/reperfusion injury and to a lesser extent the calcium paradox involves the generation of oxygen free radicals. The source of oxygen free radical generation in the calcium paradox is perhaps less obvious than in the oxygen paradox. It is proposed that during calcium-free perfusion, calcium is leached from the plasmalemma of the myocyte. There is a resulting increase in membrane fluidity. Within the plasmalemma are a number of calcium sensitive phospholipases. Upon reperfusion with a calcium replete medium, calcium could pool around these membrane bound phospholipases initiating a chain reaction of lipid peroxidation which actually is perpetuated by free radical generation (Equations 5A-5C). Lipid peroxidation opens channels within the plasmalemma rendering a 'leaky' sarcolemma. It is through these channels that calcium could flow down its concentration gradient into the cell. The increased calcium accumulation at the mitochondria would lead to an uncoupling of oxidative phosphorylation. With depleted energy stores, the mitochondria and sarcoplasmic reticulum no longer serve as calcium sinks. This would contribute to the calcium overload seen upon reperfusion. The role of oxygen free radical production would appear to occur during the hypoxic phase of the oxygen paradox and the ischemic phase of ischemic/reperfusion injury and during the reoxygenation/reperfusion phases. With the onset of hypoxia and/or myocardial ischemia there is an increase in reducing equivalents, disturbance and dissociation of intramitochondrial electron transport and release of ubisemiquinone, flavoproteins and superoxide radicals. The increase in reducing equivalents includes NADPH and, in ischemia, catecholamines, hypoxanthine and an increase on xanthine oxidase activity. All of these substrates are capable of participating in free radical production. This increase in free radical production in ischemic tissue is enhanced by acidosis which in the ischemic and hypoxic myocardium approaches pH 6.0-6.4.(ABSTRACT TRUNCATED AT 400 WORDS)     

Antiarrhythmic effect of ischemic preconditioning during low-flow ischemia

Abstract. Short episodes of ischemia (ischemic preconditioning) protect the heart against ventricular arrhythmias during zero-flow ischemia and reperfusion. However, in clinics, many episodes of ischemia present a residual flow (low-flow ischemia). Here we examined whether ischemic preconditioning protects against ventricular arrhythmias during and after a low-flow ischemia and, if so, by what mechanism(s).Isolated rat hearts were subjected to 60 min of low-flow ischemia (12% residual coronary flow) followed by 60 min of reperfusion. Ischemic preconditioning was induced by two cycles of 5 min of zero-flow ischemia followed by 5 and 15 min of reperfusion, respectively. Arrhythmias were evaluated as numbers of ventricular premature beats (VPBs) as well as incidences of ventricular tachycardia (VT) and ventricular .brillation (VF) during low-flow ischemia and reperfusion. Ischemic preconditioning significantly reduced the number of VPBs and the incidence of VT and of VF during low-flow ischemia. This antiarrhythmic effect of preconditioning was abolished by HOE 140 (100 nM), a bradykinin B₂ receptor blocker. Similar to preconditioning, exogenous bradykinin (10 nM) reduced the number of VPBs and the incidence of VT and of VF during low-flow ischemia. Furthermore, the antiarrhythmic effects of both ischemic preconditioning and bradykinin were abolished by glibenclamide (1 µM), a non-specific blocker of ATP-sensitive K⁺ (K_ATP) channels. Finally, the antiarrhythmic effects of both ischemic preconditioning and bradykinin were abolished by HMR 1098 (10 µM), a sarcolemmal K_ATP channel blocker but not by 5-hydroxydecanoate (100 µM), a mitochondrial K_ATP channel blocker. In conclusion, ischemic preconditioning protects against ventricular arrhythmias induced by low-flow ischemia, and this protection involves activation of bradykinin B₂ receptors and subsequent opening of sarcolemmal but not of mitochondrial K_ATP channels.     

Diazoxide对乳鼠窦房结细胞模拟缺血-再灌注时的保护作用研究

目的 :探讨K+ ATP通道开放剂diazoxide对模拟缺血 再灌注 (I/R)时培养乳鼠窦房结细胞的保护作用及其可能机制。方法 :分离乳鼠窦房结细胞 ,纯化培养 2d后进行实验。随机分为对照组、模拟I/R组、dia zoxide干预 (D +I/R)组及K+ ATP通道阻断剂 5 HD干预——— 5 HD +D +I/R组及 5 HD +I/R组。以流式细胞术检测各组窦房结细胞存活率 ;用激光共聚焦显微镜测定各组窦房结细胞内Ca2 + 。并采用全细胞膜片钳技术测定各组细胞L型钙电流 (L ICa)密度。结果 :①D +I/R组窦房结细胞存活率 [(6 1.4 3± 5 .14 ) % ]较I/R组 [(5 1.79±6 .2 8) % ]增加 (P <0 .0 1) ;5 HD +D +I/R组 [(5 2 .35± 4 .94 ) % ]及 5 HD +I/R组 [(5 3.16± 5 .35 ) % ]明显增高 ,均P <0 .0 1;②D +I/R组窦房结细胞相对荧光值较I/R组、5 HD +D +I/R组及 5 HD +I/R显著降低 ,均P <0 .0 1;③D +I/R组窦房结细胞L ICa密度较I/R组及 5 HD +D +I/R组明显增加 ,均P <0 .0 1。结论 :diazoxide降低窦房结细胞内钙负荷 ,对模拟I/R时的培养乳鼠窦房结细胞有保护作用 ,并可对抗模拟I/R对培养乳鼠窦房结细胞L ICa的影响 ,该作用可能与细胞线粒体K+ ATP通道的开放有关。     

Generation of metabolic oscillations by mitoKATP and ATP synthase during simulated ischemia in ventricular myocytes

Metabolic oscillations and the concomitant periodic activations of sarcolemmal ATP-sensitive K(+) channels (sarcK(ATP)) have recently been proposed as one mechanism underlying ischemia-related arrhythmia. In this study, we investigated the role of mitochondrial ATP-sensitive K(+) channels (mitoK(ATP)) and ATP synthase in the generation of metabolic oscillations during simulated ischemia from rat ventricular myocytes using patch-clamp technique and fluorescence microscopy. We have found that the combined application of creatine kinase (CK) inhibitor, 2,4-dinitrofluorobenzene, with cyanide, electron-transport-chain inhibitor causes oscillatory activations of sarcK(ATP). The oscillatory activations of sarcK(ATP) were accompanied by large periodic depolarizations in mitochondrial membrane potential (Psi(m)). 5-Hydroxydecanoate, an inhibitor of mitoK(ATP), halted the oscillations in Psi(m) at repolarized state, whereas oligomycin, an inhibitor of ATP synthase, halted them at depolarized state. In both conditions, oscillatory activations of sarcK(ATP) were abolished. Inhibitors of adenine nucleotide translocator and permeability transition pore had no effect on the oscillations in Psi(m) and sarcK(ATP). 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, an inhibitor of mitochondrial inner-membrane anion channel (IMAC), caused a full depolarization in Psi(m) and activation of sarcK(ATP), finally resulting in irreversible hypercontracture. Taken together, oscillations in Psi(m) can be explained by balance between depolarizing power of mitoK(ATP) and repolarizing power of the reverse activity of ATP synthase. ATP consumption by ATP synthase in reverse mode links periodic depolarizations in Psi(m) to oscillatory activation of sarcK(ATP). Considering that such oscillations were not induced by cyanide alone, CK system may act as an important buffer, inhibiting arrhythmia during ischemia.     

Mechanisms of cardioprotective effects of magnesium on hypoxia-reoxygenation-induced injury

During cardiac ischemia or hypoxia, increased levels of extracellular Mg show cardioprotective effects. The mechanisms of high level Mg-induced cardioprotection were examined in Langendorff perfused rat hearts. In the control group (1.2 mM Mg during hypoxia), the recovery of the left ventricular developed pressure (LVDP) after 30 min of reoxygenation was 57.6±3.0% of the level observed before hypoxia. In the high Mg group (12 mM Mg during hypoxia), the time course of recovery was faster than in the control group; the recovery level of LVDP improved to 78.4±4.2%. This protective effect of high levels of Mg decreased to 69.0±3.6% with the application of 5-hydroxydecanoic acid (100 μM), a specific mitochondrial ATP-sensitive potassium channel (K_ATP) blocker. In the low Ca group (0.2 mM Ca during hypoxia), the recovery of LVDP did not reach the level observed in the high Mg group (64.7±5.9%), but with application of diazoxide, a specific mitochondrial K_ATP channel opener, the LVDP recovery improved to 81.8±11.1%, similar to the level observed in the high Mg group. These results suggest that cardioprotective effects of high levels of extracellular Mg during hypoxia occur not only due to energy conservation and/or by intracellular prevention of Ca²⁺ over-load, but also by opening of the mitochondrial K_ATP channel.     

活性氧簇在肝脏移植缺血再灌注中的作用

肝移植缺血再灌注是一个复杂的、多因子参与的病理生理过程,包括活性氧、细胞因子、枯否细胞和中性粒细胞的激活．氧化应激是许多肝病的主要发病机制,在肝脏移植中是缺血再灌注引起肝损伤的主要原因．氧分子和活性氧簇(reactive oxygen species,ROS)的化学、生理功能,以及促氧化物和抗氧化物之间的平衡对正常的线粒体和细胞功能是至关重要的． ROS的主要来源是肝脏中的线粒体和细胞色素P450酶,以及库氏细胞和中性粒细胞,其在缺血再灌注和缺血预处理过程中对损伤的决定性作用存在争议．     

Acetylcholine-induced production of reactive oxygen species in adult rabbit ventricular myocytes is dependent on phosphatidylinositol 3- and Src-kinase activation and mitochondrial K(ATP) channel opening

Acetylcholine (ACh), like ischemic preconditioning (PC), protects against infarction and is dependent on generation of reactive oxygen species (ROS). To investigate the mechanism by which ACh causes ROS production, isolated adult rabbit cardiomyocytes underwent a timed incubation in reduced MitoTracker Red, which is oxidized to a fluorescent form after exposure to ROS. The mitochondrial ATP-sensitive potassium (mK(ATP)) channel opener diazoxide (50 microM) increased fluorescence by 47 +/- 9% (P = 0.007), indicating that opening of mK(ATP) leads to ROS generation, and that increase was blocked by the mK(ATP) blocker 5-hydroxydecanoate (5HD, 1 mM); 250 microM ACh caused a similar increase in ROS generation (+45 +/- 6% for all experiments, P < 0.001). ACh-induced ROS production was prevented by (1) blockade of muscarinic surface receptors with 100 microM atropine (-6 +/- 2%, P = n.s.) or 250 nM 4-DAMP (+5 +/- 13%, P = n.s.), indicating that ACh's effect was receptor mediated; (2) closing K(ATP) channels with either the non-selective channel closer glibenclamide (50 microM) (-1.2 +/- 17%, P = n.s.) or the selective mK(ATP) closer 5HD (-1.8 +/- 9%, P = n.s.), indicating that increased ROS production involved opening of mK(ATP); (3) blockade of mitochondrial electron transport chain with 200 nM myxothiazol (-4 +/- 9%, P = n.s.), indicating ROS came from the mitochondria; (4) addition of 100 nM wortmannin (-13 +/- 12%, P = n.s.), indicating that phosphatidylinositol 3-(PI3)-kinase was involved; and (5) blockade of Src-kinase with 1 microM PP2 (-2 +/- 5%, P = n.s.), indicating the involvement of an Src-kinase. These results support the hypothesis that occupation of muscarinic surface receptors by ACh causes activation of PI3- and Src-kinases that then open mK(ATP) resulting in mitochondrial ROS generation and triggering of the preconditioned state.     

Ischemic preconditioning protects hepatocytes via reactive oxygen species derived from Kupffer cells in rats

BACKGROUND AND AIMS: Hepatic ischemic preconditioning decreases sinusoidal endothelial cell injury and Kupffer cell activation after cold ischemia/reperfusion, leading to improved survival of liver transplant recipients in rats. Ischemic preconditioning also protects livers against warm ischemia/reperfusion injury, in which hepatocyte injury is remarkable. We aimed to determine whether ischemic preconditioning directly protects hepatocytes and to elucidate its mechanisms. METHODS: Rats were injected with gadolinium chloride to deplete Kupffer cells or with N -acetyl- l -cysteine, superoxide dismutase, or catalase to scavenge reactive oxygen species. Livers were then preconditioned by 10 minutes of ischemia and 10 minutes of reperfusion. Subsequently, livers were subjected to 40 minutes of warm ischemia and 60 minutes of reperfusion in vivo or in a liver perfusion system. In other rats, livers were preconditioned by H(2)O(2) perfusion instead of ischemia. In the other experiments, livers were perfused with nitro blue tetrazolium to detect reactive oxygen species formation. RESULTS: Ischemic preconditioning decreased injury in hepatocytes, but not in sinusoidal endothelial cells. Kupffer cell depletion itself did not change hepatocyte injury after ischemia/reperfusion, indicating no contribution of Kupffer cells to ischemia/reperfusion injury. However, Kupffer cell depletion reversed hepatoprotection by ischemic preconditioning. Reactive oxygen species formation occurred in Kupffer cells after ischemic preconditioning. Scavenging of reactive oxygen species reversed the effect of ischemic preconditioning, and H(2)O(2) preconditioning mimicked ischemic preconditioning. CONCLUSIONS: Ischemic preconditioning directly protected hepatocytes after warm ischemia/reperfusion, which is not via suppression of changes in sinusoidal cells as in cold ischemia/reperfusion injury. This hepatocyte protection was mediated by reactive oxygen species produced by Kupffer cells.     

Myocardial recovery from global ischemia and reperfusion: Effects of pre- and/or post-ischemic perfusion with low-Ca2+

The effects of brief (5 min) pre- and/or post-ischemic treatment with low-Ca²⁺ (10^?4m) on cardiac mechanical performance during and after increasing periods of total global ischemia were investigated on isolated perfused rat heart. Mechanical parameters studied were changes in diastolic resting length (ΔDRL), ventricular contraction amplitude, its first derivative $\text{d}\text{L}\text{d}\text{t}{}_{\mathrm{<mtext>max</mtext>}}$, and cardiac responsiveness to variations in extracellular Ca²⁺. The results demonstrate that pre-ischemic low-Ca²⁺ may completely prevent: (a) the development of myocardial contracture during 45 min ischemia, (b) the occurrence of post-ischemic contracture and (c) a loss in myocardial contractile activity after 30 min of total ischemia. Post-ischemic low-Ca²⁺ was clearly less effective in protecting the heart against ischemia-induced loss in contractile function and offered no additional benefit when combined with pre-ischemic low-Ca²⁺. Post-ischemic mechanical recovery appeared to correlate better with (end)-reperfusion-contracture than with (end)-ischemic contracture. Cardiac responsiveness to perfusate-Ca²⁺ decreased with increasing duration of the ischemic period (>30 min), but was not significantly altered by the low-Ca²⁺-treatments. It was concluded from the results that the protective effect of a pre-ischemic low-Ca²⁺ treatment is superior to that of a similar post-ischemic treatment. The mechanism of protection and the role of Ca²⁺ in the ischemic process are discussed in terms of changes in the availability of intracellular free-Ca²⁺.     

Modulation of cardiac interstitial noradrenaline levels through KATP channels during ischemic preconditioning in rabbits: comparison of the effect of anesthesia between pentobarbital and ketamine + xylazine

Summary In rabbits, both the stimulation of α1-adrenoceptors and ischemic preconditioning (PC) reduce infarct size. One candidate for the mechanism of PC is noradrenaline (NA), which stimulates α1-adrenoceptors in the myocardium during PC. Opening of the K_ATP channel is considered to be another candidate for PC, since a K_ATP channel blocker, glibenclamide, blocks the infarct size-reducing effect of the PC of 5-min ischemia and 5-min reperfusion in rabbits anesthetized with ketamine + xylazine. However, in rabbits anesthetized with pentobarbital, the infarct size-reducing effect of PC was not blocked by glibenclamide. The effect of glibenclamide on the PC effect thus differs depending on the anesthesia used. Therefore, we speculated that the increase in cardiac interstitial NA levels induced by PC may be modified by the anesthesia used, thus regulating the effect of glibenclamide on the PC effect. In open-chest Japanese white male rabbits anesthetized with pentobarbital or ketamine + xylazine, myocardial interstitial NA levels were measured before and during the PC of 5-min ischemia and 5-min reperfusion in the presence or absence of the K_ATP channel blocker, glibenclamide (0.3mg/kg, i.v.), using a microdialysis technique. The NA levels were measured using high-performance liquid chromatography coupled with electrochemical detection. The PC of 5-min ischemia and 5-min reperfusion significantly elevated the interstitial NA level. This increase in the NA level was not blocked by glibenclamide under anesthesia with pentobarbital. Under anesthesia with ketamine + xylazine, the PC did not cause an increase in the myocardial interstitial NA level in either the absence or the presence of glibenclamide. In conclusion, PC elevates the myocardial interstitial NA level, and this elevation is not mediated through the opening of the K_ATP channel under anesthesia with pentobarbital. Under anesthesia with ketamine + xylazine, PC does not cause an increase in the myocardial interstitial NA level. This may explain the discrepancy in the blocking effect of glibenclamide on the infarct size-reducing effect of PC between anesthesia with pentobarbital and ketamine + xylazine.     

Oxygen-mediated myocardial damage during ischaemia and reperfusion: role of the cellular defences against oxygen toxicity

The possibility that myocardial ischaemia alters the defence mechanisms against oxygen toxicity has been investigated. Ischaemia was induced in isolated, perfused rabbit hearts by reducing coronary flow from 25 ml/min to 1 ml/min for 90 min. Two different degrees of ischaemic damage have been achieved using either spontaneously beating or electrically stimulated hearts. The effects of post-ischaemic reperfusion were also followed for 30 min. Tissue activity of superoxide dismutase (SOD), glutathione peroxidase and reductase (GPD and GRD) have been determined together with tissue content of reduced and oxidized glutathione (GSH and GSSG) and of protein SH groups. The changes in myocardial ATP and CP content and release of CPK and of GSH and GSSG were also determined. Systolic and diastolic pressures were continuously monitored. In the spontaneously beating hearts ischaemia induced a reduction of tissue GSH and protein SH groups. On reperfusion there was a recovery of mechanical function, a transient release of GSH into the coronary effluent and an increase of tissue GSH. In the paced hearts, ischaemia resulted in 50% reduction of mitochondrial SOD activity together with a reduction of tissue GSH and protein SH groups. Reperfusion induced a massive release of CPK and of GSH and GSSG, a further reduction of tissue GSH concomitant with an increase of GSSG and no recovery of mechanical function. GPD and GRD activity were not affected by ischaemia and reperfusion. These data indicate that severe ischaemia induces a reduction of the protective mechanisms against oxygen toxicity.     

The role of cardiac mitochondria in the regulation of intracellular calcium during ischemia and reperfusion: X-ray microanalysis using freeze-dried sections

Summary The calcium concentration in papillary muscles was measured by X-ray microanalysis in order to clarify the role played by mitochondria in intracellular calcium regulation during ischemia and reperfusion. Rat hearts perfused by the Langendorff method were rapidly frozen prior to and during ischemia, as well as following reperfusion. Sections prepared by cryoultramicrotomy were freeze-dried, carbon-coated, and analyzed in an electron microscope. A new freeze-drying procedure was developed, in which the ultrastructure was well-preserved, with sarcomeres, triads, and mitochondria easily recognized. Calcium accumulation into the mitochondria occurred during 30-min ischemia (29.7 ± 17.0 mmol/kg dry weight) and increased further after 15-min reperfusion (157.1 ± 104.5), the calcium concentration decreased after 60-min reperfusion (58.1 ± 29.0). However, the calcium concentration in the cytosol did not change significantly. It is thought that mitochondrial calcium accumulation is reversible, to a certain degree, and that the mitochondria play a part in intracellular calcium regulation in pathological states.     

Inhibition of phenylephrine induced hypertrophy in rat neonatal cardiomyocytes by the mitochondrial KATP channel opener diazoxide

The effect of the putative mitochondrial K(ATP) channel opener diazoxide (100 microM) was studied in terms of its ability to modulate the hypertrophic effect of 24 h treatment with the alpha(1) adrenoceptor agonist phenylephrine (PE; 10 microM) in cultured neonatal rat ventricular myocytes. PE on its own significantly increased cell size by 40%, (3)H leucine incorporation by 37% and produced more than a threefold elevation in both atrial natriuretic peptide and myosin light chain-2 expression. These effects were nearly completely prevented by diazoxide although the inhibitory effect of this agent was generally mitigated by the mitochondrial K(ATP) channel antagonists 5-hydroxydecanoic acid (100 microM) and glibenclamide (50 microM). Although PE produced an early threefold elevation in MAP kinase activation this was generally unaffected by diazoxide. PE also produced a greater than threefold increase in Na-H exchanger isoform 1 (NHE-1) expression which, was prevented by diazoxide treatment. Our study therefore, demonstrates a potential antihypertrophic influence of mitochondrial K(ATP) channel activation which, is related to diminished NHE-1 expression. Mitochondrial K(ATP) channel activation could represent an effective approach to minimize the myocardial hypertrophic process.     

Effects of CCCP-induced mitochondrial uncoupling and cyclosporin A on cell volume,cell injury and preconditioning protection of isolated rabbit cardiomyocytes

Cell swelling may contribute to acute cell injury subsequent to ischemia/reperfusion. The potential role of mitochondrial uncoupling and the resultant mitochondrial swelling, due to opening of the mitochondrial permeability transition pore (MPTP), were examined in an in vitro ischemically pelleted isolated rabbit cardiomyocyte model using the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP) to uncouple mitochondria. Cyclosporin A (CsA) was employed to inhibit MPTP opening. Cell volume was determined by a cell-flotation, density-gradient assay, using bromododecane. Cell viability, subsequent to an osmotic stress, was determined by trypan blue permeability. Ischemic preconditioning (IPC) facilitated volume regulation following an osmotic stress. Ischemic-cell swelling was reduced by IPC. IPC protected ischemically pelleted cells, but CsA had no significant effects on injury or IPC protection. CCCP ischemia accelerated rates of ischemic contracture and injury, and abolished IPC protection. IPC protection was restored by CsA. In CCCP-ischemic-uncoupled cells, subjected to a reduced (170 mOsm) osmotic stress, CsA and IPC afforded independent and additive protection. Chelerythrine and 5-hydroxydecanoate (5-HD) blocked IPC, but did not reduce CsA protection. Electron microscopy confirmed that CCCP ischemia induced mitochondrial matrix swelling that was reduced by CsA. Cardioprotection by IPC and CsA was accompanied by proportional reductions in cell swelling. Morphometric analysis of the electron photomicrographs demonstrated that the mitochondrial volume fractions were significantly reduced in the CsA/CCCP (29.8 +/- 2.3%, P < 0.004) and IPC/CsA/CCCP (31.5 +/- 1.7%, P < 0.0008) groups as compared to the CCCP-ischemic group (40.5 +/- 1.7%) The IPC/CCCP group (39.5 +/- 4.2%) was not significantly different from the CCCP-ischemic group. NIM 811, a CsA analogue MPTP blocker with no calcineurin inhibitory activity, afforded protection similar to CsA. The results suggest that CsA protection may, in part, be mediated by reduction of mitochondrial swelling.     

Beneficial effects of adenosine triphosphate-sensitive K+ channel opener on liver ischemia/reperfusion injury

AIM: To investigate the effect of diazoxide administration on liver ischemia/reperfusion injury.METHODS: Wistar male rats underwent partial liver ischemia performed by clamping the pedicle from the medium and left anterior lateral segments for 1 h under mechanical ventilation. They were divided into 3 groups: Control Group, rats submitted to liver manipulation, Saline Group, rats received saline, and Diazoxide Group, rats received intravenous injection diazoxide (3.5 mg/kg) 15 min before liver reperfusion. 4 h and 24 h after reperfusion, blood was collected for determination of aspartate transaminase (AST), alanine transaminase (ALT), tumor necrosis factor (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10), nitrite/nitrate, creatinine and tumor growth factor-β1 (TGF-β1). Liver tissues were assembled for mitochondrial oxidation and phosphorylation, malondialdehyde (MDA) content, and histologic analysis. Pulmonary vascular permeability and myeloperoxidase (MPO) were also determined.RESULTS: Four hours after reperfusion the diazoxide group presented with significant reduction of AST (2009 ± 257 U/L vs 3523 ± 424 U/L, P = 0.005); ALT (1794 ± 295 U/L vs 3316 ± 413 U/L, P = 0.005); TNF-α (17 ± 9 pg/mL vs 152 ± 43 pg/mL, P = 0.013; IL-6 (62 ± 18 pg/mL vs 281 ± 92 pg/mL); IL-10 (40 ± 9 pg/mL vs 78 ± 10 pg/mL P = 0.03), and nitrite/nitrate (3.8 ± 0.9 μmol/L vs 10.2 ± 2.4 μmol/L, P = 0.025) when compared to the saline group. A significant reduction in liver mitochondrial dysfunction was observed in the diazoxide group compared to the saline group (P < 0.05). No differences in liver MDA content, serum creatinine, pulmonary vascular permeability and MPO activity were observed between groups. Twenty four hours after reperfusion the diazoxide group showed a reduction of AST (495 ± 78 U/L vs 978 ± 192 U/L, P = 0.032); ALT (335 ± 59 U/L vs 742 ± 182 U/L, P = 0.048), and TGF-β1 (11 ± 1 ng/mL vs 17 ± 0.5 ng/mL, P = 0.004) serum levels when compared to the saline group. The control group did not present alterations when compared to the diazoxide and saline groups.CONCLUSION: Diazoxide maintains liver mitochondrial function, increases liver tolerance to ischemia/reperfusion injury, and reduces the systemic inflammatory response. These effects require further evaluation for using in a clinical setting.     

Contractile failure in early myocardial ischemia: Models and mechanisms

Summary In early myocardial ischemia we find a number of salient electrical and ionic alterations. This article reviews action potential shortening, K accumulation, and contractile failure. Enhanced K efflux during early myocardial ischemia has been attributed to a number of mechanisms, including: the inhibition of active K uptake, osmotic changes, efflux of K ions linked to anion extrusion, cation exchange, altered cellular energy levels, in particular, the opening of ATP-dependent K channels, the involvement of other ion channels, a H/K-ion exchanger, and a catecholamine-dependent pathway. The different mechanisms are discussed. Action potential shortening was described as a salient characteristic of myocardial ischemia in 1954 by Trautwein and Dudel, and was attributed to enhanced outward current. Recently it has been shown by several authors that ATP-dependent potassium channels play a key role in this context. Contractile failure in early myocardial ischemia has been explained by shortening of the action potential duration, reduced cytoplasmic free calcium levels, intracellular acidification, and a rise in inorganic phosphate and Mg. In summary, it is concluded that ATP-dependent K channels may be involved in each of these three phenomena.     

Endogenous glycogen prevents Ca2+ overload and hypercontracture in harp seal myocardial cells during simulated ischemia

The purpose of this study was to determine if elevated myocardial glycogen content could obviate Ca(2+) overload and subsequent myocardial injury in the setting of low oxygen and diminished exogenous substrate supplies. Isolated harp seal cardiomyocytes, recognized as having large glycogen stores, were incubated under conditions simulating ischemia (oxygen and substrate deprivation) for 1 h. Rat cardiomyocytes were used for comparison. Freshly isolated seal cardiomyocytes contained approximately 10 times more glycogen than those from rats (479 +/- 39 vs. 48 +/- 5 nmol glucose/mg dry weight (dry wt), mean +/- S.E., n = 6), and during ischemia lactate production was significantly greater in seal compared to rat cardiomyocytes (660 +/- 99 vs. 97 +/- 14 nmol/mg dry wt), while glycogen content decreased both in seal (from 479 +/- 39 to 315 +/- 58 nmol glucose/mg dry wt) and rat cardiomyocytes (from 48 +/- 5 to 18 +/- 5 nmol glucose/mg dry wt). Cellular ATP was well maintained in ischemic seal cardiomyocytes, whereas it showed a 65% decline (from 31 +/- 3 to 11 +/- 1 nmol ATP/mg dry wt) in rat cardiomyocytes. Similarly, total seal cardiomyocyte Ca(2+) content was not affected by ischemia, while Ca(2+) increased from 8.5 +/- 2.0 to 13.3 +/- 2.0 nmol/mg dry wt in ischemic rat myocytes. Rat cardiomyocytes also showed a notable decline in the percentage of rod-shaped cells in response to ischemia (from 66 +/- 4% to 30 +/- 3%), and cell morphology was unaffected in seal incubations. Addition of iodoacetate (IAA, an inhibitor of glycolysis) to seal cardiomyocytes, on top of substrate and oxygen deprivation, reduced the cellular content of ATP by 52.9 +/- 4.4% (from 25 +/- 4 to 11 +/- 2 nmol ATP/mg dry wt) and the percentage of rod-shaped myocytes from 51 +/- 3% to 28 +/- 4%, while total Ca(2+) content was unchanged by these conditions. Seal cardiomyocytes thus tolerate low oxygen conditions better than rat cardiomyocytes. This finding is most likely due to a higher glycolysis rate in seals, fueled by larger myocardial glycogen stores.