Blockade of electron transport during ischemia protects cardiac mitochondria

Subsarcolemmal mitochondria sustain progressive damage during myocardial ischemia. Ischemia decreases the content of the mitochondrial phospholipid cardiolipin accompanied by a decrease in cytochrome c content and a diminished rate of oxidation through cytochrome oxidase. We propose that during ischemia mitochondria produce reactive oxygen species at sites in the electron transport chain proximal to cytochrome oxidase that contribute to the ischemic damage. Isolated, perfused rabbit hearts were treated with rotenone, an irreversible inhibitor of complex I in the proximal electron transport chain, immediately before ischemia. Rotenone pretreatment preserved the contents of cardiolipin and cytochrome c measured after 45 min of ischemia. The rate of oxidation through cytochrome oxidase also was improved in rotenone-treated hearts. Inhibition of the electron transport chain during ischemia lessens damage to mitochondria. Rotenone treatment of isolated subsarcolemmal mitochondria decreased the production of reactive oxygen species during the oxidation of complex I substrates. Thus, the limitation of electron flow during ischemia preserves cardiolipin content, cytochrome c content, and the rate of oxidation through cytochrome oxidase. The mitochondrial electron transport chain contributes to ischemic mitochondrial damage that in turn augments myocyte injury during subsequent reperfusion.     

Studies on hepatic injury and antioxidant enzyme activities in rat subcellular organelles following in vivo ischemia and reperfusion

The activities of rat hepatic subcellular antioxidant enzymes were studied during hepatic ischemia/reperfusion. Ischemia was induced for 30 min (reversible ischemia) or 60 min (irreversible ischemia). Ischemia was followed by 2 or 24 h of reperfusion. Hepatocyte peroxisomal catalase enzyme activity decreased during 60 min of ischemia and declined further during reperfusion. Peroxisomes of normal density (d = 1.225 gram/ml) were observed in control tissues. However, 60 min of ischemia also produced a second peak of catalase specific activity in subcellular fractions corresponding to newly formed low density immature peroxisomes (d = 1.12 gram/ml). The second peak was also detectable after 30 min of ischemia followed by reperfusion for 2 or 24 h. Mitochondrial and microsomal fractions responded differently. MnSOD activity in mitochondria and microsomal fractions increased significantly (p < 0.05) after 30 min of ischemia, but decreased below control values following 60 min of ischemia and remained lower during reperfusion at 2 and 24 h in both organelle fractions. Conversely, mitochondrial and microsomal glutathione peroxidase (GPx) activity increased significantly (p < 0.001) after 60 min of ischemia and was sustained during 24 h of reperfusion. In the cytosolic fraction, a significant increase in CuZnSOD activity was noted following reperfusion in animals subjected to 30 min of ischemia, but 60 min of ischemia and 24 h of reperfusion resulted in decreased CuZnSOD activity. These studies suggest that the antioxidant enzymes of various subcellular compartments respond to ischemia/reperfusion in an organelle or compartment specific manner and that the regulation of antioxidant enzyme activity in peroxisomes may differ from that in mitochondria and microsomes. The compartmentalized changes in hepatic antioxidant enzyme activity may be crucial determinant of cell survival and function during ischemia/reperfusion. Finally, a progressive decline in the level of hepatic reduced glutathione (GSH) and concomitant increase in serum glutamate pyruvate transaminase (SGPT) activity also suggest that greater tissue damage and impairment of intracellular antioxidant activity occur with longer ischemia periods, and during reperfusion.     

Mitochondrial ATP-Sensitive K<Superscript>+</Superscript> Channels Prevent Oxidative Stress,Permeability Transition and Cell Death

Ischemia followed by reperfusion results in impairment of cellular and mitochondrial functionality due to opening of mitochondrial permeability transition pores. On the other hand, activation of mitochondrial ATP-sensitive K⁺ channels (mitoK_ATP) protects the heart against ischemic damage. This study examined the effects of mitoK_ATP and mitochondrial permeability transition on isolated rat heart mitochondria and cardiac cells submitted to simulated ischemia and reperfusion (cyanide/aglycemia). Both mitoK_ATP opening, using diazoxide, and the prevention of mitochondrial permeability transition, using cyclosporin A, protected against cellular damage, without additive effects. MitoK_ATP opening in isolated rat heart mitochondria slightly decreased Ca²⁺ uptake and prevented mitochondrial reactive oxygen species production, most notably in the presence of added Ca²⁺. In ischemic cells, diazoxide decreased ROS generation during cyanide/aglycemia while cyclosporin A prevented oxidative stress only during simulated reperfusion. Collectively, these studies indicate that opening mitoK_ATP prevents cellular death under conditions of ischemia/reperfusion by decreasing mitochondrial reactive oxygen species release secondary to Ca²⁺ uptake, inhibiting mitochondrial permeability transition.     

Attenuation of intestinal ischemia/reperfusion injury with sodium nitroprusside: studies on mitochondrial function and lipid changes

Reactive oxygen species have been implicated in cellular injury during ischemia/reperfusion (I/R). Mitochondria are one of the main targets of oxygen free radicals and damage to this organelle leads to cell death. Reports suggest that nitric oxide (NO) may offer protection from damage during I/R. This study has looked at the functional changes and lipid alteration to mitochondria during intestinal I/R and the protection offered by NO. It was observed that I/R of the intestine is associated with functional alterations in the mitochondria as suggested by MTT reduction, respiratory control ratio and mitochondrial swelling. Mitochondrial lipid changes suggestive of activation of phospholipase A(2) and phospholipase D were also seen after (I/R) mediated injury. These changes were prevented by the simultaneous presence of a NO donor in the lumen of the intestine. These studies have suggested that structural and functional alterations of mitochondria are prominent features of I/R injury to the intestine which can be ameliorated by NO.     

A computational model of cardiomyocyte metabolism predicts unique reperfusion protocols capable of reducing cell damage during ischemia/reperfusion

If a coronary blood vessel is occluded and the neighboring cardiomyocytes deprived of oxygen, subsequent reperfusion of the ischemic tissue can lead to oxidative damage due to excessive generation of reactive oxygen species. Cardiomyocytes and their mitochondria are the main energy producers and consumers of the heart, and their metabolic changes during ischemia seem to be a key driver of reperfusion injury. Here, we hypothesized that tracking changes in cardiomyocyte metabolism, such as oxygen and ATP concentrations, would help in identifying points of metabolic failure during ischemia and reperfusion. To track some of these changes continuously from the onset of ischemia through reperfusion, we developed a system of differential equations representing the chemical reactions involved in the production and consumption of 67 molecular species. This model was validated and used to identify conditions present during periods of critical transition in ischemia and reperfusion that could lead to oxidative damage. These simulations identified a range of oxygen concentrations that lead to reverse mitochondrial electron transport at complex I of the respiratory chain and a spike in mitochondrial membrane potential, which are key suspects in the generation of reactive oxygen species at the onset of reperfusion. Our model predicts that a short initial reperfusion treatment with reduced oxygen content (5% of physiological levels) could reduce the cellular damage from both of these mechanisms. This model should serve as an open-source platform to test ideas for treatment of the ischemia reperfusion process by following the temporal evolution of molecular concentrations in the cardiomyocyte.     

Metabolic stages, mitochondria and calcium in hypoxic/ischemic brain damage

Cerebral hypoxia/ischemia leads to mitochondrial dysfunction due to lack of oxygen leaving the glycolytic metabolism as a main pathway for ATP production. Inhibition of mitochondrial respiration thus triggers generation of lactate and hydrogen ions (H+), and furthermore dramatically reduces ATP generation leading to disregulation of cellular ion metabolism with subsequent intracellular calcium accumulation. Upon reperfusion, when mitochondrial dysfunction is (at least partially) reversed by restoring cerebral oxygen supply, bioenergetic metabolism recovers and brain cells are able to re-institute their normal ionic homeostatic mechanisms. However, the initial restoration of normal mitochondrial function may be only transient and followed by a secondary, delayed perturbation of mitochondrial respiratory performance seen as a decrease in cellular ATP levels and known as "secondary energy failure". There have been several mechanisms considered responsible for delayed post-ischemic mitochondrial failure, the mitochondrial permeability transition (MPT) being one that is considered important. Although the amount of calcium available during early reperfusion in vivo is limited, relative to the amount needed to trigger the MPT in vitro; the additional intracellular conditions (of acidosis, high phosphate, and low adenine nucleotideae levels) prevailing during reperfusion, favor MPT pore opening in vivo. Furthermore, the cellular redistribution and/or changes in the intracellular levels of pro-apoptotic proteins can alter mitochondrial function and initiate apoptotic cell death. Thus, mitochondria seem play an important role in orchestrating cell death mechanisms following hypoxia/ischemia. However, it is still not clear which are the key mechanisms that cause mitochondrial dysfunction and lead ultimately to cell death, and which have more secondary nature to brain damage acting as aggravating factors.     

ATP hydrolysis by ischemic mitochondria

Cellular ATP levels are determined by the rates of ATP production and ATP hydrolysis. Both phenomena are affected by ischemia. Mitochondrial enzymes are damaged, inhibiting this organelle's ability to make ATP. Mitochondria are also uncoupled by ischemia and have the ability to hydrolyze ATP. We designed a series of experiments to determine whether decreased production or increased hydrolysis of ATP was the primary effect of mitochondrial damage. Rat hearts were subjected to 45 min of warm ischemia in order to induce irreversible cell damage. ATP or ADP was injected into cuvettes containing mitochondria isolated from normal myocardium or myocardium damaged by ischemia. Luciferin-luciferase, which fluoresces in the presence of ATP, was also added to the tubes as an indicator of ATP levels. Mixtures of uncoupled and coupled mitochondria were made and compared with the mitochondria damaged by ischemia. The results showed that mitochondria damaged by prolonged ischemia hydrolyze ATP more rapidly than normal mitochondria; however, normal mitochondria can easily compensate for increased ATP hydrolysis when in mixture with equal amounts of uncoupled mitochondria. These data suggests that the low cellular levels of ATP following irreversible ischemia are primarily due to decreased ATP synthesis and not to increased hydrolysis.     

Ischemia, rather than reperfusion, inhibits respiration through cytochrome oxidase in the isolated, perfused rabbit heart: role of cardiolipin

Ischemia and reperfusion result in mitochondrial dysfunction, with decreases in oxidative capacity, loss of cytochrome c, and generation of reactive oxygen species. During ischemia of the isolated perfused rabbit heart, subsarcolemmal mitochondria, located beneath the plasma membrane, sustain a loss of the phospholipid cardiolipin, with decreases in oxidative metabolism through cytochrome oxidase and the loss of cytochrome c. We asked whether additional injury to the distal electron chain involving cardiolipin with loss of cytochrome c and cytochrome oxidase occurs during reperfusion. Reperfusion did not lead to additional damage in the distal electron transport chain. Oxidation through cytochrome oxidase and the content of cytochrome c did not further decrease during reperfusion. Thus injury to cardiolipin, cytochrome c, and cytochrome oxidase occurs during ischemia rather than during reperfusion. The ischemic injury leads to persistent defects in oxidative function during the early reperfusion period. The decrease in cardiolipin content accompanied by persistent decrements in the content of cytochrome c and oxidation through cytochrome oxidase is a potential mechanism of additional myocyte injury during reperfusion.     

Isolating the segment of the mitochondrial electron transport chain responsible for mitochondrial damage during cardiac ischemia

Ischemia damages the mitochondrial electron transport chain (ETC), mediated in part by damage generated by the mitochondria themselves. Mitochondrial damage resulting from ischemia, in turn, leads to cardiac injury during reperfusion. The goal of the present study was to localize the segment of the ETC that produces the ischemic mitochondrial damage. We tested if blockade of the proximal ETC at complex I differed from blockade distal in the chain at cytochrome oxidase. Isolated rabbit hearts were perfused for 15 min followed by 30 min stop-flow ischemia at 37 °C. Amobarbital (2.5 mM) or azide (5 mM) was used to block proximal (complex I) or distal (cytochrome oxidase) sites in the ETC. Time control hearts were buffer-perfused for 45 min. Subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) were isolated. Ischemia decreased cytochrome c content in SSM but not in IFM compared to time control. Blockade of electron transport at complex I preserved the cytochrome c content in SSM. In contrast, blockade of electron transport at cytochrome oxidase with azide did not retain cytochrome c in SSM during ischemia. Since blockade of electron transport at complex III also prevented cytochrome c loss during ischemia, the specific site that elicits mitochondrial damage during ischemia is likely located in the segment between complex III and cytochrome oxidase.     

Ischemic defects in the electron transport chain increase the production of reactive oxygen species from isolated rat heart mitochondria

Cardiac ischemia decreases complex III activity, cytochrome c content, and respiration through cytochrome oxidase in subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM). The reversible blockade of electron transport with amobarbital during ischemia protects mitochondrial respiration and decreases myocardial injury during reperfusion. These findings support that mitochondrial damage occurs during ischemia and contributes to myocardial injury during reperfusion. The current study addressed whether ischemic damage to the electron transport chain (ETC) increased the net production of reactive oxygen species (ROS) from mitochondria. SSM and IFM were isolated from 6-mo-old Fisher 344 rat hearts following 25 min global ischemia or following 40 min of perfusion alone as controls. H(2)O(2) release from SSM and IFM was measured using the amplex red assay. With glutamate as a complex I substrate, the net production of H(2)O(2) was increased by 178 +/- 14% and 179 +/- 17% in SSM and IFM (n = 9), respectively, following ischemia compared with controls (n = 8). With succinate as substrate in the presence of rotenone, H(2)O(2) increased by 272 +/- 22% and 171 +/- 21% in SSM and IFM, respectively, after ischemia. Inhibitors of electron transport were used to assess maximal ROS production. Inhibition of complex I with rotenone increased H(2)O(2) production by 179 +/- 24% and 155 +/- 14% in SSM and IFM, respectively, following ischemia. Ischemia also increased the antimycin A-stimulated production of H(2)O(2) from complex III. Thus ischemic damage to the ETC increased both the capacity and the net production of H(2)O(2) from complex I and complex III and sets the stage for an increase in ROS production during reperfusion as a mechanism of cardiac injury.     

Modulation of electron transport protects cardiac mitochondria and decreases myocardial injury during ischemia and reperfusion

Mitochondria are increasingly recognized as lynchpins in the evolution of cardiac injury during ischemia and reperfusion. This review addresses the emerging concept that modulation of mitochondrial respiration during and immediately following an episode of ischemia can attenuate the extent of myocardial injury. The blockade of electron transport and the partial uncoupling of respiration are two mechanisms whereby manipulation of mitochondrial metabolism during ischemia decreases cardiac injury. Although protection by inhibition of electron transport or uncoupling of respiration initially appears to be counterintuitive, the continuation of mitochondrial oxidative phosphorylation in the pathological milieu of ischemia generates reactive oxygen species, mitochondrial calcium overload, and the release of cytochrome c. The initial target of these deleterious mitochondrial-driven processes is the mitochondria themselves. Consequences to the cardiomyocyte, in turn, include oxidative damage, the onset of mitochondrial permeability transition, and activation of apoptotic cascades, all favoring cardiomyocyte death. Ischemia-induced mitochondrial damage carried forward into reperfusion further amplifies these mechanisms of mitochondrial-driven myocyte injury. Interruption of mitochondrial respiration during early reperfusion by pharmacologic blockade of electron transport or even recurrent hypoxia or brief ischemia paradoxically decreases cardiac injury. It increasingly appears that the cardioprotective paradigms of ischemic preconditioning and postconditioning utilize modulation of mitochondrial oxidative metabolism as a key effector mechanism. The initially counterintuitive approach to inhibit mitochondrial respiration provides a new cardioprotective paradigm to decrease cellular injury during both ischemia and reperfusion. cardiolipin; cytochrome c; complex I; cytochrome oxidase     

Ischemic-reperfused isolated working mouse hearts: membrane damage and type IIA phospholipase A2

For the murine heart the relationships between ischemia-reperfusion-induced loss of cardiac function, enzyme release, high-energy phosphate (HEP), and membrane phospholipid metabolism are ill-defined. Accordingly, isolated ejecting murine hearts were subjected to varying periods of ischemia, whether or not followed by reperfusion. On reperfusion, hemodynamic function was almost completely restored after 10 min of ischemia [83 +/- 14% recovery of cardiac output (CO)], but was severely depressed after 15 and 20 min of ischemia (40 +/- 24 and 31 +/- 24% recovery of CO, respectively). Reperfusion was associated with partial recovery of HEP stores and enhanced degradation of phospholipids as indicated by the accumulation of fatty acids (FA). Myocardial FA content and enzyme release during reperfusion were correlated (r = 0.70), suggesting that membrane phospholipid degradation and cellular damage are closely related phenomena. To investigate the role of type IIA secretory phospholipase A2 (sPLA2) in this process, hearts from wild-type and sPLA2-deficient mice were subjected to ischemia-reperfusion. Postischemic functional recovery, ATP depletion, enzyme release, and FA accumulation were not significantly different between wild-type and sPLA2- deficient hearts. These findings argue against a prominent role of type IIA sPLA2 in the development of irreversible cell damage in the ischemic-reperfused murine myocardium.     

Lipid peroxidation and alterations to oxidative metabolism in mitochondria isolated from rat heart subjected to ischemia and reperfusion.

Ischemia-reperfusion injury to cardiac myocytes involves membrane damage mediated by oxygen free radicals. Lipid peroxidation is considered a major mechanism of oxygen free radical toxicity in reperfused heart. Mitochondrial respiration is an important source of these reactive oxygen species and hence a potential contributor to reperfusion injury. We have examined the effects of ischemia (30 min) and ischemia followed by reperfusion (15 min) of rat hearts, on the kinetic parameters of cytochrome c oxidase, on the respiratory activities and on the phospholipid composition in isolated mitochondria. Mitochondrial content of malonyldialdheyde (MDA), an index of lipid peroxidation, was also measured. Reperfusion was accompanied by a significant increase in MDA production. Mitochondrial preparations from control, ischemic and reperfused rat heart had equivalent Km values for cytochrome c, although the maximal activity of the oxidase was 25 and 51% less in ischemic and reperfused mitochondria than that of controls. These changes in the cytochrome c oxidase activity were associated to parallel changes in state 3 mitochondrial respiration. The cytochrome aa3 content was practically the same in these three types of mitochondria. Alterations were found in the mitochondrial content of the major phospholipid classes, the most pronounced change occurring in the cardiolipin, the level that decreased by 28 and by 50% as function of ischemia and reperfusion, respectively. The lower cytochrome c oxidase activity in mitochondria from reperfused rat hearts could be almost completely restored to the level of control hearts by exogenously added cardiolipin, but not by other phospholipids nor by peroxidized cardiolipin. It is proposed that the reperfusion-induced decline in the mitochondrial cytochrome c oxidase activity can be ascribed, at least in part, to a loss of cardiolipin content, due to peroxidative attack of its unsaturated fatty acids by oxygen free radicals. These findings may provide an explanation for some of the factors that lead to myocardial reperfusion injury.     

Increased state 4 mitochondrial respiration and swelling in early post-ischemic reperfusion of rat heart

Isolated rat hearts were exposed to 30 min ischemia or to 30 min ischemia followed by 2, 5 or 40 min reperfusion and mitochondria were isolated at these different time points. ADP-stimulated, succinate-dependent respiration rate (state 3) was not significantly changed at the different time points examined. In contrast, state 4 (non-ADP-stimulated) respiration rate was significantly increased after 30 min ischemia, and it increased further during the first post-ischemic reperfusion period. Mitochondrial swelling, as evaluated under conditions of the major controlled ion channels (i.e. permeability transition pore and ATP-dependent mitochondrial K(+) channel) closed, significantly increased in parallel. It is suggested that the inner mitochondrial membrane permeability is increased under exposure of the heart to ischemia and early reperfusion, and that the phenomenon is reversible upon subsequent long periods of reperfusion.     

Ischemic preconditioning attenuates ischemia-reperfusion-induced mucosal apoptosis by inhibiting the mitochondria-dependent pathway in rat small intestine

Ischemic preconditioning provides a way of protecting organs from damage inflicted with prolonged ischemia-reperfusion. In this study, we investigated the mechanism of ischemic preconditioning involved in inhibition of prolonged ischemia-reperfusion-induced mucosal apoptosis in rat small intestine. Ischemic preconditioning was triggered by a transient occlusion of the superior mesenteric artery followed by reperfusion. Ischemia-reperfusion was induced by 60-min occlusion of the superior mesenteric artery followed by 60-min reperfusion in the small intestine. Ischemia-reperfusion alone induced mucosal apoptosis and mitochondrial respiratory dysfunction via promoted reactive oxygen species generation, reduced mitochondrial glutathione oxidation, increased mitochondrial lipid peroxidation, reduced mitochondrial membrane potential, and enhanced release of cytochrome c from mitochondria to activate caspase-9 and caspase-6 in the small intestine. Pretreatment with 20-min ischemia followed by 5-min reperfusion significantly inhibited the prolonged ischemia-reperfusion-induced mucosal apoptosis by 30%. Ischemic preconditioning ameliorated mitochondrial respiratory dysfunction by 50%, reduced reactive oxygen species generation by 38%, and suppressed mitochondrial lipid peroxidation by 36%, resulting in improvement of the mitochondrial membrane potential and prevention of cytochrome c release as well as caspase-6 activation. Results suggest that ischemic preconditioning attenuated ischemia-reperfusion-induced mucosal apoptosis partly by inhibiting the reactive oxygen species-mediated mitochondria-dependent pathway in the rat small intestine.     

Parallel changes in brain tissue blood flow and mitochondrial function during and after 30 minutes of bilateral forebrain ischemia in the gerbil

Forebrain ischemia was induced in Mongolian gerbils by bilateral occlusion of the common carotid arteries for 30 minutes. These animals do not have a complete circulus arteriosus Willisii. Mitochondria were prepared from the forebrain tissue at the end of the 30 minutes occlusion period as well as at different time points after the release of the occlusion. Tissue blood flow in the forebrain was also determined by measuring the brain tissue accumulation of 14C-iodoantipyrine. Tissue blood flow in the forebrain decreased from a control level of 1.43 +/- 0.03 ml/min/gr to 0.13 +/- 0.03 ml/min/gr by the 30th minute of ischemia, increased to 1.12 +/- 0.25 ml/min/gr after 5 minutes of reflow, but decreased again to 0.41 +/- 0.07 ml/min/gr after 1 1/2 hours of reflow. Oxygen consumption rate of mitochondria prepared from the forebrain (glutamate + malate as substrates in the presence of ADP) was 98 +/- 13 nmoles O2/min/mg protein in control animals, decreased to 61 +/- 9 nmoles O2/min/mg protein after 30 minutes of occlusion, recovered to 106 +/- 9 nmoles O2/min/mg protein during the first 30 minutes of reperfusion. During extended reperfusion, mitochondrial respiratory activity declined reaching 20 +/- 5 nmoles O2/min/mg protein after 5 1/2 hours of reperfusion. Respiratory control ratio of the mitochondria (relative increase of respiration upon addition of ADP) was 9.2 +/- 1.3 in control animals, 7.0 +/- 1.5 after 30 minutes of carotid occlusion, 9.0 +/- 1.2 after 30 minutes of reperfusion, and 5.8 +/- 0.8 after 5 1/2 hours of reperfusion. Superoxide dismutase activity of the forebrain mitochondria was 5.10 +/- 0.7 I.U./mg protein in control animals, decreased to 3.3 +/- 1.6 I.U./mg protein after 30 minutes of occlusion and remained at this level throughout the reperfusion period. These data confirm earlier reports that deterioration of mitochondrial function may contribute to the development of ischemic and post-ischemic brain tissue damage. It also appears possible that postischemic damage of mitochondrial function develops secondary to postischemic deterioration of tissue blood flow.     

Generation of superoxide anion by mitochondria and impairment of their functions during anoxia and reoxygenation in vitro

A small portion of the oxygen consumed by aerobic cells is converted to superoxide anion at the level of the mitochondrial respiratory chain. If produced in excess, this harmful radical is considered to impair cellular structures and functions. Damage at the level of mitochondria have been reported after ischemia and reperfusion of organs. However, the complexity of the in vivo system prevents from understanding and describing precise mechanisms and locations of mitochondrial impairment. An in vitro model of isolated-mitochondria anoxia-reoxygenation is used to investigate superoxide anion generation together with specific damage at the level of mitochondrial oxidative phosphorylation. Superoxide anion is detected by electron paramagnetic resonance spin trapping with POBN-ethanol. Mitochondrial respiratory parameters are calculated from oxygen consumption traces recorded with a Clark electrode. Respiring mitochondria produce superoxide anion in unstressed conditions, however, the production is raised during postanoxic reoxygenation. Several respiratory parameters are impaired after reoxygenation, as shown by decreases of phosphorylating and uncoupled respiration rates and of ADP/O ratio and by increase of resting respiration. Partial protection of mitochondrial function by POBN suggests that functional damage is related and secondary to superoxide anion production by the mitochondria in vitro.     

Aging selectively decreases oxidative capacity in rat heart interfibrillar mitochondria

Mitochondrial-derived oxidative injury contributes to cellular aging as well as to reperfusion-induced tissue damage. While the aging-heart suffers greater tissue damage following ischemia and reperfusion than the adult heart, the occurrence of aging-related alterations in mitochondrial oxidative metabolism in the elderly heart has remained uncertain. We determined if aging altered oxidative metabolism in either of the two populations of cardiac mitochondria, subsarcolemmal mitochondria (SSM) that reside beneath the plasma membrane or interfibrillar mitochondria (IFM) located between the myofibrils. SSM and IFM were isolated from 6-month adult and 24- and 28-month elderly Fischer 344 rat hearts. Aging-related alterations were limited to IFM, while SSM remained unaffected. Aging decreased the rate of oxidative phosphorylation in IFM, including when stimulated by electron donors specific for cytochrome oxidase. Cytochrome oxidase enzyme activity was decreased in IFM from aging hearts, while activity in SSM remained similar to adult controls. These findings allow future studies of aging-related decrements in oxidative function to focus upon IFM, while SSM provide an inherent control group of mitochondria that are free of aging-related alterations in oxidative function. The selective alteration of IFM during aging raises the possibility that the consequences of aging-induced mitochondrial dysfunction will be enhanced in specific subcellular regions of the senescent myocyte.     

Membrane alterations in acute myocardial ischemia

The molecular consequences of acute myocardial ischemia induced in rabbit hearts by ligation of the left circumflex branch of the coronary artery were assessed in terms of the biochemical properties of subcellular organelles. Mitochondrial alteration, as reflected in progressive decrease in the activity of azide-sensitive ATPase, was apparent as early as 5 min postligation, but the activity of another mitochondrial enzyme, cytochrome c oxidase, was unchanged, even following 60 min of coronary ligation. Sarcolemmal Na+K+-ATPase exhibited a time course of inactivation similar to that of the mitochondrial ATPase, but differed from the latter in that the impairment was not reversed on reperfusion. Cellular levels of ATP, which decreased in parallel with the loss of ATPase activities, also remained depressed following reperfusion. Decreases in lysosomal enzyme latency were noted, but these occurred somewhat later than the sarcolemmal and mitochondrial alterations. Attempts to demonstrate the production of a population of labile lysosomal structures during ischemia were unsuccessful. Similarly, no alterations in the gel electrophoretic profiles of proteins or in the P phosphatidylcholine/P phosphatidylethanolamine ratio of isolated mitochondrial or sarcolemmal membranes from hearts subjected to ischemia and (or) subsequent reperfusion could be found. It is suggested that sarcolemmal Na+,K+-ATPase may serve as a sensitive and readily quantifiable index of irreversible cellular necrosis and, therefore, be of value in assessing the possible beneficial effects of pharmacological interventions.     

Endothelial NOS activity and myocardial oxygen metabolism define the salvageable ischemic time window for ischemic postconditioning

Ischemic postconditioning (IPOC) could be ineffective or even detrimental if the index ischemic duration is either too short or too long. The present study is to demonstrate that oxygen supply and metabolism defines a salvageable ischemic time window of IPOC in mice. C57BL/6 mice underwent coronary artery occlusion followed by reperfusion (I/R), with or without IPOC by three cycles of 10 s/10 s R/I. In vivo myocardial tissue oxygenation was monitored with electron paramagnetic resonance oximetry. Regional blood flow (RBF) was measured with a laser Doppler monitor. At the end of 60 min reperfusion, tissue from the risk area was collected, and mitochondrial enzyme activities were assayed. Tissue oximetry demonstrated that I/R induced a reperfusion hyperoxygenation state in the 30- and 45-min but not 15- and 60-min ischemia groups. IPOC attenuated the hyperoxygenation with 45 but not 30 min ischemia. RBF, eNOS phosphorylation, and mitochondrial enzyme activities were suppressed after I/R with different ischemic time, and IPOC afforded protection with 30 and 45 but not 60 min ischemia. Infarct size measurement indicated that IPOC reduced infarction with 30 and 45 min but not 60 min ischemia. Clearly, IPOC protected mouse heart with a defined ischemic time window between 30 and 45 min. This salvageable time window was accompanied by the improvement of RBF due to increased phosphorylated eNOS and the preservation of mitochondrial oxygen consumption due to conserved mitochondrial enzyme activities. Interestingly, this salvageable ischemic time window was mirrored by tissue hyperoxygenation status in the postischemic heart.