Mitochondrial damage during ischemia determines post-ischemic contractile dysfunction in perfused rat heart

Possible mechanisms underlying sodium overload-induced ischemia/reperfusion injury in perfused rat hearts were examined. Massive accumulation of myocardial Na(+) occurred during ischemia, suggesting cytosolic sodium overload in cardiac cells. Treatment of the pre-ischemic heart with 0.3 micromol/l tetrodotoxin or 3 micromol/l ethyl-isopropyl amiloride enhanced post-ischemic contractile recovery (72 or 82% of initial vs 24% for untreated group), which was associated with suppression of tissue Na(+) accumulation (138 or 141% of initial vs 270% for untreated group), restoration of tissue high-energy phosphates, and preservation of the ability of mitochondria to produce ATP in the ischemic/reperfused heart. The release of cytochrome c from the ischemic heart was observed, which was blocked by treatment of the pre-ischemic heart with these agents. The improvement of post-ischemic contractile recovery by these agents was closely correlated with the ability of mitochondria to produce ATP during ischemia. To examine the effects of sodium overload on mitochondrial function, isolated mitochondria were incubated in the presence of various concentrations of Na(+). Na(+) induced mitochondrial membrane perturbations such as depolarization of the membrane potential, mitochondrial swelling, cytochrome c release from isolated mitochondria, and a reduction in oxidative phosphorylation. These events in the isolated mitochondria were not blocked by the presence of the above agents. The results suggest that cytosolic sodium overload in cardiac cells may induce deterioration of the mitochondrial function during ischemia and that this mitochondrial damage may determine post-ischemic contractile dysfunction in perfused rat hearts.     

Effects of N-(2-mercaptopropionyl)-glycine on mitochondrial function in ischemic-reperfused heart

OBJECTIVE: A possible mechanism for N-(2-mercaptopropionyl)-glycine (MPG) underlying the improvement of contractile function and mitochondrial activity of ischemic-reperfused rat hearts was examined. METHODS: Isolated, perfused hearts were subjected to 35 min ischemia-60 min reperfusion. At the end of ischemia or reperfusion, myocardial Na(+) content and mitochondrial oxygen consumption rate (OCR) were examined. The perfused heart was treated with 0.1-1 mM MPG for 30 min prior to ischemia or for the first 30 min of reperfusion. RESULTS: Ischemia increased myocardial Na(+) content (sodium overload) and decreased mitochondrial OCR. The left ventricular developed pressure (LVDP) of the untreated heart recovered to 19.8+/-3.8% of the preischemic value and the infarct area amounted to 23.3+/-1.7% of the left ventricle. The thiobarbiturate-reacting substance (TRS) was also increased in the reperfused, but not ischemic, myocardium. Pretreatment of the perfused heart with 0.3-1 mM MPG attenuated the ischemia-induced sodium overload and decrease in the OCR. Pretreatment with the agent also enhanced the postischemic recovery of LVDP, attenuated reperfusion-induced increase in TRS, and reduced the infarct area. Although the postischemic treatment with MPG suppressed the increase in TRS in the reperfused myocardium, a LVDP recovery of reperfused hearts was not observed. Cardiac mitochondria were isolated and examined for the direct effect of MPG on their function. Incubation with either 12.5 mM sodium lactate or 1 microM phenylarsine oxide neither altered the mitochondrial membrane potential nor induced mitochondrial swelling, whereas incubation with a combination of these agents elicited the membrane potential depolarization and swelling. Incubation of mitochondria with 1 mM MPG attenuated these events. CONCLUSION: These results suggest that both attenuation of sodium overload and preservation of the mitochondrial function may largely contribute to cardioprotection of MPG in the ischemic-reperfused heart.     

Calcium-free reperfusion prevents mitochondrial calcium accumulation but exacerbates injury

Excessive accumulation of intracellular calcium has been proposed as a mediator of cell injury during ischemia and reperfusion. To test whether restriction of extracellular calcium might ameliorate hepatic injury following hypothermic ischemic preservation, isolated perfused rat livers were reperfused with a medium with normal extracellular calcium (mM-Ca) or no added calcium (microM-Ca) following 26 hr preservation at 4 degrees C. During reperfusion mitochondria from mM-Ca livers accumulated calcium and respiratory activity declined. Although mitochondrial calcium accumulation was prevented by microM-Ca reperfusion, damage to respiratory activity was exacerbated. Edema formation and loss of gluconeogenesis were similarly exacerbated in microM-Ca. This accelerated damage was not reversed by subsequent restoration of calcium. These results demonstrate that rather than ameliorating cell injury, calcium deprivation during reperfusion exacerbates damage to mitochondrial and cellular functions. Thus, cellular or mitochondrial calcium accumulation does not appear to be a sine qua non for hepatocyte injury during reperfusion following hypothermic ischemia.     

Postischaemic reperfusion injury in the isolated rat heart: effect of ruthenium red.

STUDY OBJECTIVE--The aim was to investigate the effect of attenuating mitochondrial calcium uptake with ruthenium red on myocardial function and the resultant necrosis following prolonged ischaemia and reperfusion in isolated rat hearts. Mitochondrial dysfunction, secondary to increased calcium uptake, has been implicated as an important mediator of reperfusion injury in the heart. DESIGN--To examine the role of mitochondrial calcium uptake in mediating ischaemic and reperfusion injury, isolated rat hearts were perfused with ruthenium red (n = 6), a polysaccharide dye which inhibits calcium uptake by mitochondria, and were compared to control perfused hearts (n = 7). After stabilisation, hearts were subjected to 60 min no flow ischaemia, immediately followed by 40 min reperfusion. EXPERIMENTAL MATERIAL--Hearts were used from male Wistar rats weighing 300-350 g. MEASUREMENTS AND MAIN RESULTS--Cardiac high energy phosphates (ATP, phosphocreatine, inorganic phosphate) and pH were continuously monitored during ischaemia and reperfusion using phosphorus magnetic resonance spectroscopy. Contractility (dP/dT), coronary flow, creatine kinase release, and the time to the onset of ischaemic contracture were also measured. No differences in metabolic abnormalities or time to peak contraction during ischaemia were found between groups, suggesting that ruthenium red does not alter the metabolic consequences of ischaemia. However, upon reperfusion, the following differences in the ruthenium red perfused hearts were observed when compared to control hearts (p less than 0.05): ATP and phosphocreatine recovery were more complete, myocardial contractility was greater, coronary flow was greater, and myocyte necrosis was attenuated. CONCLUSIONS--Combined with the known inhibitory effect of ruthenium red on mitochondrial calcium uptake, these data suggest that an important component of myocardial injury following ischaemia and reperfusion in the isolated rat heart is the result of mitochondrial calcium accumulation.     

Fasting in vivo delays myocardial cell damage after brief periods of ischemia in the isolated working rat heart

To assess the effects of fasting on recovery of function and exogenous glucose metabolism after 15 minutes of total ischemia, we perfused isolated working rat hearts from fed and fasted animals. Hearts were perfused in a recirculating system with bicarbonate buffer containing glucose (10 mM). Mechanical performance, release of marker proteins for ischemic membrane damage (lactate dehydrogenase, myoglobin, citrate synthase), and the concentrations of lactate and glucose in the perfusion medium were measured serially. Tissue metabolites were also measured. Fasting raised the myocardial glycogen content by 25%. Cardiac performance of perfused hearts from fed and fasted animals was the same during the preischemic and the post-ischemic period. The time of return of function to preischemic values was significantly less in hearts from fasted rats (2.3 versus 7.8 minutes, p less than 0.025). The release of cytosolic and mitochondrial marker proteins was significantly lower in hearts from fasted rats than in hearts from fed rats. Glucose metabolic rates during control and reperfusion were unchanged for hearts from fasted rats, but decreased for hearts from fed rats during reperfusion. The adenine nucleotide content at the end of ischemia was higher in hearts from fasted animals than in hearts from fed animals. We conclude that increasing glycogen levels prior to ischemia improves recovery of function, lessens membrane damage, and prevents loss of adenine nucleotides.     

Inhibition of Na+-H+ exchanger protects diabetic and non-diabetic hearts from ischemic injury: insight into altered susceptibility of diabetic hearts to ischemic injury.

It has been previously suggested that alterations in sodium homeostasis, leading to calcium overload may play a part in the mediation of cardiac ischemic injury. It has been demonstrated that the Na+-H+ exchanger plays an important role with regard to the regulation of intracellular sodium during ischemia and reperfusion and that inhibition of the Na+-H+ exchanger during ischemia protects hearts from ischemic injury. Studies using chemically-induced diabetic animals have suggested that the cardiac Na+-H+ exchanger in the diabetic heart is impaired and is responsible for limiting the increase in sodium during ischemia. The extent to which the Na+-H+ exchanger contributes to increases in intracellular sodium during ischemia in diabetic hearts is unclear as direct measurements of exchanger activity have not been made in genetically diabetic hearts. Therefore, this paper aims to address the following issues: (a) is the Na+-H+ exchanger impaired in a genetically diabetic rat heart: (b) does this impairment result in lower [Na]i or [Ca]i during ischemia; and (c) does Na+-H+ exchanger inhibition limit injury and functional impairment in diabetic hearts during ischemia and reperfusion? These issues were examined by inhibiting the Na+-H+ exchanger with ethylisopropylamiloride (EIPA) in isolated perfused hearts from both genetically diabetic (BB/W) and non-diabetic rats. Levels of intracellular sodium, intracellular calcium, intracellular pH and high energy phosphates (using 23Na,19F, 31P NMR spectroscopies, respectively) during global ischemia and reperfusion were also measured. The impact of diabetes on Na+-H+ exchanger activity was assessed by measuring pH recovery of these hearts after an acid load. Creatine kinase release during reperfusion was used as a measure of ischemic injury. This study demonstrated that the Na+-H+ exchanger is impaired in diabetic hearts. Despite this impaired activity, inhibition of Na+-H+ exchanger protected diabetic hearts from ischemic injury and was associated with attenuation of the rise in sodium and calcium, and limitation of acidosis and preservation of ATP during ischemia. The data presented here favor the use of Na+-H+ exchanger inhibitors to protect ischemic myocardium in diabetics. Also, the data provides possible mechanisms for the altered susceptibility of diabetic hearts to ischemic injury.     

Decrease in mitochondrial complex I activity in ischemic/reperfused rat heart: involvement of reactive oxygen species and cardiolipin

Reactive oxygen species (ROS) are considered an important factor in ischemia/reperfusion injury to cardiac myocytes. Mitochondrial respiration is an important source of ROS production and hence a potential contributor to cardiac reperfusion injury. In this study, we have examined the effect of ischemia and ischemia followed by reperfusion of rat hearts on various parameters related to mitochondrial function, such as complex I activity, oxygen consumption, ROS production, and cardiolipin content. The activity of complex I was reduced by 25% and 48% in mitochondria isolated from ischemic and reperfused rat heart, respectively, compared with the controls. These changes in complex I activity were associated with parallel changes in state 3 respiration. The capacity of mitochondria to produce H2O2 increased on reperfusion. The mitochondrial content of cardiolipin, which is required for optimal activity of complex I, decreased by 28% and 50% as function of ischemia and reperfusion, respectively. The lower complex I activity in mitochondria from reperfused rat heart could be completely restored to the level of normal heart by exogenous added cardiolipin. This effect of cardiolipin could not be replaced by other phospholipids nor by peroxidized cardiolipin. It is proposed that the defect in complex I activity in ischemic/reperfused rat heart could be ascribed to a ROS-induced cardiolipin damage. These findings may provide an explanation for some of the factors responsible for myocardial reperfusion injury.     

缺血后处理减轻心肌线粒体损伤对缝隙连接蛋白43的影响

目的观察缺血后处理对线粒体缝隙连接蛋白43(Cx43)的影响以及心肌保护的可能机制。方法健康新西兰大白兔64只.建立心肌缺血再灌注模型,随机分为4组,假手术组、缺血再灌注组、缺血预处理组、缺血后处理组,每组16只。检测各组心肌梗死面积,透射电镜观察心肌细胞的超微结构,荧光法检测线粒体膜电位,比色法检测线粒体Ca~(2+)和丙二醛浓度及超氧化物歧化酶(SOD)活性,Western blot法检测Cx43含量。结果与缺血再灌注组比较,缺血后处理组和缺血预处理组兔心肌梗死面积明显减少,心肌线粒体形态结构改变明显减轻,跨膜电位、SOD活性、线粒体Cx43明显升高,Ca~(2+)、丙二醛浓度明显降低(P<0.05,P<0.01)。与假手术组比较,缺血再灌注组兔线粒体Cx43明显下调(P<0.05)。结论缺血后处理保护心肌及线粒体可能与提高线粒体跨膜电位、降低线粒体氧自由基水平和减轻线粒体Ca~(2-)超载有关,其机制可能与提高线粒体Cx43表达有关。     

Cardioprotection and altered mitochondrial adenine nucleotide transport

It is becoming increasingly clear that mitochondrial dysfunction is critically important in myocardial ischemic injury, and that cardioprotective mechanisms must ultimately prevent or attenuate mitochondrial damage. Mitochondria are also essential for energy production, and therefore prevention of mitochondrial injury must not compromise oxidative phosphorylation during reperfusion. This review will focus on one mitochondrial mechanism of cardioprotection involving inhibition of adenine nucleotide transport across the outer mitochondria membrane under de-energized conditions. This slows ATP hydrolysis by the mitochondria, and would be expected to lower mitochondrial membrane potential during ischemia, to inhibit calcium uptake during ischemia, and potentially to reduce free radical generation during early reperfusion. Two interventions that similarly inhibit mitochondrial adenine nucleotide transport are Bcl-2 overexpression and GSK inhibition. A possible final common mechanism shared by both of these interventions is discussed.     

Prevention of transcoronary macromolecular leakage after ischemia-reperfusion by the calcium entry blocker nisoldipine. Direct observations in isolated rat hearts

Coronary microvascular damage appears to play a role in reperfusion injury after myocardial ischemia. This study was designed to afford direct viewing of the effects of myocardial ischemia-reperfusion on the coronary microcirculation and to determine whether pretreatment with the calcium blocker nisoldipine would attenuate any microvascular damage during reperfusion. Four groups of isolated rat hearts were perfused with a solution that contained red cells and fluorescent albumin, but was essentially free of platelets and leukocytes. Group I served as a nonischemic control. Group II hearts were subjected to 30 minutes of no-flow ischemia followed by reperfusion. Group III hearts were pretreated with nisoldipine (1 microgram/min) for 5 minutes before ischemia, and group IV hearts were treated with nitroglycerin (93 micrograms/min) before and after ischemia to mimic the vasodilation caused by nisoldipine. Perfused coronary capillarity and transcoronary extravasation of plasma albumin were measured by direct visualization techniques before and after ischemia. For group I, there was no significant change in coronary resistance, perfused capillarity, or transcoronary extravasation with time. For both groups II and IV, ischemia-reperfusion caused no increase in coronary resistance, but a significant decrease in perfused capillarity and a marked increase in transcoronary extravasation of fluorescent albumin (P less than 0.05). The nisoldipine group (group III) demonstrated a similar decrease in perfused capillarity but no increase in protein extravasation during reperfusion. These results indicate that, in the heart, platelets and/or leukocytes are not absolutely necessary to induce either the no-reflow phenomenon or the permeability damage observed during reperfusion after ischemia. The protective effect of treatment with nisoldipine appeared to be independent of vasodilation. We speculate that this calcium blocker reduced endothelial uptake of calcium during reperfusion, preventing endothelial deformation and formation of interendothelial gaps.     

Intracellular free calcium and mitochondrial membrane potential in ischemia/reperfusion and preconditioning

Moderation of calcium perturbations has been implicated in ischemic preconditioning. As mitochondria possess an effective Ca(2+)transporting system driven by the mitochondrial membrane potential, experiments were performed to study time-averaged intracellular free calcium and the mitochondrial membrane potential during preconditioning and ischemia-reperfusion. Isolated rat hearts were subjected to 5 min of preconditioning, a 9-min intervening reperfusion and 21 min of ischemia with subsequent reperfusion. The hearts were preloaded with the Ca(2+)indicator Fura-2 or the mitochondrial membrane potential probe safranine. A method was devised for correction for NADH autofluorescence in time-averaged Ca(2+)probing with Fura-2. The pH dependence of the apparent dissociation constant of the Ca(2+)complex of Fura-2 was determined. Intracellular free Ca(2+)increased during the 5-min ischemia, and this was reversed upon reperfusion. During protracted ischemia a continual Ca(2+)rise was observed when the fluorescence data were corrected for changes in pH. An initial sharp Fura-2 fluorescence spike upon final reperfusion was caused by a pH-dependent change in the dissociation constant of the Ca(2+)complex of Fura-2. In preconditioned hearts the free Ca(2+)was somewhat lower during reperfusion, but a major effect of preconditioning was observed during the prolonged ischemia. The decrease in mitochondrial membrane potential during prolonged ischemia was faster in the preconditioned heart with no difference during the final reperfusion. The effect of preconditioning on cell survival was reflected in a decrease in the post-ischemic washout of creatine kinase. The moderation of the ischemic and post-ischemic intracellular Ca(2+)increase, and the acceleration of the ischemic mitochondrial membrane potential decrease by ischemic preconditioning is in accord with the notion of the involvement of mitochondrial ATP sensitive K(+)channels in preconditioning. In studies on ischemia it is absolutely necessary to correct for the pH-sensitivity of the apparent dissociation constant of the calcium complex of Fura-2 to obtain reliable data for intracellular free calcium.     

Cardiac reperfusion injury: Aging, lipid peroxidation, and mitochondrial dysfunction

Cardiac reperfusion and aging are associated with increased rates of mitochondrial free radical production. Mitochondria are therefore a likely site of reperfusion-induced oxidative damage, the severity of which may increase with age. 4-Hydroxy-2-nonenal (HNE), a major product of lipid peroxidation, increases in concentration upon reperfusion of ischemic cardiac tissue, can react with and inactivate enzymes, and inhibits mitochondrial respiration in vitro. HNE modification of mitochondrial protein(s) might, therefore, be expected to occur during reperfusion and result in loss in mitochondrial function. In addition, this process may be more prevalent in aged animals. To begin to test this hypothesis, hearts from 8- and 24-month-old rats were perfused in Langendorff fashion and subjected to periods of ischemia and/or reperfusion. The rate of state 3 respiration of mitochondria isolated from hearts exposed to ischemia (25 min) was approximately 25% less than that of controls, independent of age. Reperfusion (40 min) caused a further decline in the rate of state 3 respiration in hearts isolated from 24- but not 8-month-old rats. Furthermore, HNE modification of mitochondrial protein (~30 and 44 kDa) occurred only during reperfusion of hearts from 24-month-old rats. Thus, HNE-modified protein was present in only those mitochondria exhibiting reperfusion-induced declines in function. These studies therefore identify mitochondria as a subcellular target of reperfusion damage and a site of age-related increases in susceptibility to injury.     

Effects of calcium antagonists and free radical scavengers on myocardial ischemia and reperfusion injury: evaluation by 31P-NMR spectroscopy

The Langendorff perfused rat heart was used to investigate whether myocardial damage during ischemia and reperfusion could be protected by free radical scavengers, calcium antagonist and adenosine. Myocardial high energy phosphates were measured by phosphorus-31 NMR spectroscopy during normal perfusion, 20 min of ischemia and 20 min of reperfusion. In hearts, which were treated both with free radical scavengers (FRS) (Superoxide dismutase): 24 IU/ml and catalase 22 IU/ml) and verapamil (10(-7) M), beta-ATP was significantly higher than that of FRS at the end of ischemia. However, beta-ATP recovered only to 83% of baseline value at the end of reperfusion. In view of myocardial metabolism, verapamil treated hearts were good for recovery of creatine phosphate (PCr) but not ATP at the end of reperfusion. Hearts which were treated with only adenosine did not differ from control hearts. However, when hearts were treated with both verapamil and adenosine (10(-4) M), recovery of both ATP and PCr content was significantly greater than that of control hearts. These results suggested that pretreatment with both verapamil and adenosine before and after global ischemia could protect ischemic myocardium, but, further studies are necessary to clarify the precise mechanism of protection.     

Glycogen synthase kinase 3 inhibition slows mitochondrial adenine nucleotide transport and regulates voltage-dependent anion channel phosphorylation

Inhibition of glycogen synthase kinase (GSK)-3 reduces ischemia/reperfusion injury by mechanisms that involve the mitochondria. The goal of this study was to explore possible molecular targets and mechanistic basis of this cardioprotective effect. In perfused rat hearts, treatment with GSK inhibitors before ischemia significantly improved recovery of function. To assess the effect of GSK inhibitors on mitochondrial function under ischemic conditions, mitochondria were isolated from rat hearts perfused with GSK inhibitors and were treated with uncoupler or cyanide or were made anoxic. GSK inhibition slowed ATP consumption under these conditions, which could be attributable to inhibition of ATP entry into the mitochondria through the voltage-dependent anion channel (VDAC) and/or adenine nucleotide transporter (ANT) or to inhibition of the F(1)F(0)-ATPase. To determine the site of the inhibitory effect on ATP consumption, we measured the conversion of ADP to AMP by adenylate kinase located in the intermembrane space. This assay requires adenine nucleotide transport across the outer but not the inner mitochondrial membrane, and we found that GSK inhibitors slow AMP production similar to their effect on ATP consumption. This suggests that GSK inhibitors are acting on outer mitochondrial membrane transport. In sonicated mitochondria, GSK inhibition had no effect on ATP consumption or AMP production. In intact mitochondria, cyclosporin A had no effect, indicating that ATP consumption is not caused by opening of the mitochondrial permeability transition pore. Because GSK is a kinase, we assessed whether protein phosphorylation might be involved. Therefore, we performed Western blot and 1D/2D gel phosphorylation site analysis using phos-tag staining to indicate proteins that had decreased phosphorylation in hearts treated with GSK inhibitors. Liquid chromatographic-mass spectrometric analysis revealed 1 of these proteins to be VDAC2. Taken together, we found that GSK-mediated signaling modulates transport through the outer membrane of the mitochondria. Both proteomics and adenine nucleotide transport data suggest that GSK regulates VDAC and that VDAC may be an important regulatory site in ischemia/reperfusion injury.     

Effects of propionyl-L-carnitine on isolated mitochondrial function in the reperfused diabetic rat heart

The effects of propionyl-L-carnitine (PLC) on isolated mitochondrial respiration in the ischemic reperfused diabetic heart were studied. Oral PLC treatment of STZ-diabetic rats was initiated for a period of 6 weeks. After treatment, isolated working hearts from diabetic rats were perfused under aerobic conditions then subjected to 25 min of no-flow ischemia followed by 15 min of aerobic reperfusion. At the end of reperfusion, heart mitochondria was isolated using differential centrifugation and respiration measured in the presence of pyruvate, glutamate, and palmitoylcarnitine. Our results indicate that diabetes was characterized by a pronounced decrease in heart function under aerobic conditions as well as during reperfusion following ischemia. Treatment with PLC resulted in a significant improvement in heart function under these conditions. The depressions in state 3 mitochondrial respiration with both pyruvate and glutamate seen in reperfused hearts from diabetic rats were prevented by PLC. State 3 respiration in the presence of palmitoylcarnitine was also improved in the ischemic reperfused diabetic rat heart. Our results show that PLC improves recovery of mechanical function following ischemia in the diabetic rat heart. The beneficial effects of PLC are associated with enhanced mitochondrial oxidation of fuels.     

Interfibrillar cardiac mitochondrial comples III defects in the aging rat heart

We used the Fischer 344 rat as a model foraging effects on the heart. Cardiac interfibrillar mitochondria (IFM), located between the myofibrils, exhibit a decrease in protein yield and oxidative phosphorylation through complex III and IV in elderly (24 months)compared to adult controls (6 months). In contrast, subsarcolemmal mitochondria (SSM)located beneath the plasma membrane remained unchanged. The activity of electron transport complex III decreased only in the IFM with aging. Complex III and IV require an inner mitochondrial membrane lipid, cardiolipin for maximal activity. However, the content and composition of cardiolipin was unchanged in the IFM from aging hearts. We observed electron leakage in complex III at the myxothiazol site in the aging IFM accompanied by increased superoxide production. The aging heart sustains greater injury during ischemia and reperfusion compared to adult hearts. We propose that ischemic damage combines with aging defects in complex III to increase oxidative injury in aging hearts. Ischemia damaged complex III in both SSM and IFM from adult and aging hearts via impairment of the iron-sulfur subunit without the loss of the apo protein. Thus, at the onset of reperfusion, 0complex III in IFM contains two defects in electron flow, which are likely to prime complex III for enhanced oxidant production during reperfusion, leading to increased damage in aging hearts. This revised version was published online in July 2006 with corrections to the Cover Date.     

Enhanced modification of cardiolipin during ischemia in the aged heart

Aging enhances cardiac injury during ischemia and reperfusion compared to the adult heart, including in the Fischer 344 rat model of aging (F344). In interfibrillar cardiac mitochondria obtained from the elderly F344 rat, the rate of oxidative phosphorylation and the activity of electron transport complex III is decreased, concomitant with an increase in the production of reactive oxygen species. In the isolated, perfused heart, 25 min of global ischemia results in additional damage to complex III. We proposed that ischemic damage superimposed upon the aging defect augments production of reactive oxygen species leading to greater oxidative damage in the aged heart. Cardiolipin is an oxidatively sensitive phospholipid located in the inner mitochondrial membrane. Oxidative damage to cardiolipin was assessed by characterization of the individual molecular species of cardiolipin via reverse phase HPLC and electrospray mass spectrometry (MS). The predominant molecular species of cardiolipin (95%) contains four linoleic acid residues (C18:2). Ischemia and reperfusion did not alter the content or composition of cardiolipin in the adult heart. Following ischemia and reperfusion in the aged heart, a new molecular species of cardiolipin was present with mass increased by 48 Da, suggesting the addition of three oxygen atoms. MS fragmentation localized the added mass to the C18:2 residues. Ischemia alone was sufficient to modify cardiolipin in the aged heart whereas cardiolipin in the adult heart remained unaltered. Thus, age-enhanced oxidative damage occurs within mitochondria in the heart during ischemia and reperfusion, especially during ischemia.     

Lysoplasmenylethanolamine accumulation in ischemic/reperfused isolated fatty acid-perfused hearts.

Lysophospholipid accumulation has been implicated in the pathogenesis of irreversible injury during myocardial ischemia and reperfusion. Plasmalogens (phospholipids with a vinyl-ether bond in the sn-1 position) account for more than 50% of total myocardial sarcolemmal and sarcoplasmic reticulum phospholipids. Accumulation of plasmalogen choline and ethanolamine lysophospholipids (lysoplasmenylcholine and lysoplasmenylethanolamine) or the effects of exogenous fatty acids on lysoplasmalogen accumulation during ischemia and reperfusion have not been examined. Isolated working rat hearts perfused with buffer containing either 11 mM glucose or 11 mM glucose plus 1.2 mM palmitate were subjected to aerobic, ischemic, or ischemia/reperfusion protocols. Levels of lysoplasmenylcholine and lysoplasmenylethanolamine were quantified using a two-stage high-performance liquid chromatographic technique. In hearts perfused with glucose alone, no significant differences in levels of lysoplasmenylcholine or lysoplasmenylethanolamine were seen during ischemia or reperfusion. In fatty acid-perfused hearts, however, significant accumulation of lysoplasmenylethanolamine occurred during reperfusion but not during ischemia (723 +/- 112, 734 +/- 83, and 1,394 +/- 193 nmol/g dry wt for aerobic, ischemic, and ischemic/reperfused hearts, respectively; p less than 0.05 for ischemic/reperfused hearts versus aerobic or ischemic hearts). Lysoplasmenylcholine levels after ischemia and reperfusion did not differ significantly from aerobic values, regardless of whether fatty acids were present or absent from the perfusate. Aerobic and ischemic/reperfused rabbit hearts, in the presence of fatty acid, showed a similar profile in their lysoplasmalogen content. We conclude that differential lysoplasmenylethanolamine accumulation occurs during myocardial reperfusion when exogenous fatty acid concentrations are high. This may reflect the selective action of fatty acid intermediates on the metabolism of lysoplasmenylethanolamines.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of TA-3090, a new calcium channel blocker, on myocardial substrate utilization in ischemic and nonischemic isolated working fatty acid-perfused rat hearts.

Experimental studies have shown that calcium channel blockade has a protective effect on the ischemic myocardium. Although these agents may act by decreasing intracellular Ca2+ accumulation during reperfusion or to reduce oxygen requirements by decreasing myocardial work load, recent evidence suggests that calcium blockers may also favorably alter energy substrate metabolism in ischemic and reperfused myocardium. In this study, TA-3090, a new calcium channel blocker with minimal effect on myocardial work load, was used to study the effect of calcium channel blockade on both myocardial substrate utilization and reperfusion recovery of ischemic hearts. Isolated working rat hearts were perfused at an 11.5 mm Hg preload and an 80 mm Hg afterload with Krebs-Henseleit buffer containing 11 mM glucose, 1.2 mM palmitate, and 500 microunits/ml insulin. In aerobically perfused spontaneously beating hearts, a 0.5 microM dose of TA-3090 had a mild depressant effect on heart rate but no effect on peak systolic pressure development. In paced hearts (250 beats/min), 0.5 microM TA-3090 had no effect on either peak systolic pressure development or contractility. Fatty acid and glucose oxidation was determined by measuring 14CO2 production in hearts perfused with either [14C]palmitate or [14C]glucose, respectively, whereas glycolysis was determined by measuring 3H2O production from [3H]glucose. Under aerobic conditions, fatty acid oxidation was not altered by TA-3090, but a significant decrease in glucose oxidation and glycolytic rates was observed. If hearts were subjected to a 30-minute period of no-flow ischemia, the addition of 0.5 microM TA-3090 to the perfusate before ischemia significantly improved reperfusion recovery of mechanical function. The protective effects of TA-3090 were not observed if TA-3090 was added at the time of reperfusion and were not related to a depression of function before ischemia. TA-3090, added before ischemia, significantly reduced glycogen and ATP depletion during no-flow ischemia and also significantly decreased glycolytic rates in hearts subjected to low-flow ischemia (coronary flow = 0.5 ml/min). Combined, our data suggest that the beneficial effects of calcium channel blockade on the ischemic myocardium are not related solely to a decrease in myocardial work load or metabolic demand before ischemia, but rather may in part be related to a decrease in myocardial energy demand during ischemia itself, resulting in preservation of ATP and a decrease in glycolysis. The decrease in glycolytic rates during ischemia may also result in a reduction of glycolytic product accumulation during ischemia.     

Intermittent ischemia produces a cumulative depletion of mitochondrial adenine nucleotides in the isolated perfused rat heart

The purpose of the present study was to determine if repetitive myocardial ischemia would result in the cumulative loss of mitochondrial adenine nucleotides. Isolated perfused rat hearts were subjected to continuous or intermittent ischemia. A single 5-minute period of continuous ischemia did not result in a significant decrease in the mitochondrial adenine nucleotide pool; a single 10-minute period of ischemia resulted in a decrease of approximately 17%. Next, the adenine nucleotide content of mitochondria from preischemic and 30-minute continuous ischemic hearts was compared with two groups of hearts undergoing intermittent ischemia (both groups receiving a total of 30 minutes of ischemia). One group received three 10-minute episodes of ischemia interrupted by 5-minute periods of reperfusion (3 x 10-minute intermittent ischemia); the other intermittent ischemic group received six 5-minute episodes of ischemia interrupted by 5-minute periods of perfusion (6 x 5-minute intermittent ischemia). The mitochondrial adenine nucleotide content (expressed as nanomoles per nanomole cytochrome a) for the preischemic and 30-minute continuous ischemic hearts was 14.7 +/- 0.6 and 8.0 +/- 0.4, respectively. The mitochondrial adenine nucleotide content of the 3 x 10-minute intermittent ischemia group (8.5 +/- 0.5) was not significantly different from the 30-minute continuous ischemic group. The mitochondrial adenine nucleotide content of the 6 x 5-minute intermittent ischemia group (11.0 +/- 0.6) was significantly larger than that of the 30-minute continuous and the 3 x 10-minute intermittent ischemia groups (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)