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Biomedical subjects

Edward J Lesnefsky

Publications and source records attributed to Edward J Lesnefsky.

18 recordsLinked to original sources
PubMed2006

Structure of cristae in cardiac mitochondria of aged rat.

Interfibrillar mitochondria (IFM) of the heart in aged Fischer 344 rats show a biochemical defect which might be reflected in their morphology. We examined by high resolution scanning electron microscopy over 5500 mitochondria to determine if a concomitant structural alteration existed. This methodology provides a means of examining mitochondrial cristae in three dimensions. Cristae of in situ subsarcolemmal mitochondria (SSM) and of IFM in both 6- and 24-month-old Fischer rats are predominantly lamelliform. When isolated, these organelles, whether of SSM or IFM origin, display enhanced heterogeneity, but they have similar crista morphology irrespective of the age of the rat. Crista configuration does not play a major role in age-related cardiac mitochondrial defects.

Aging↗
PubMed2006

Reversible blockade of electron transport during ischemia protects mitochondria and decreases myocardial injury following reperfusion.

Cardiac mitochondria sustain damage during ischemia and reperfusion, contributing to cell death. The reversible blockade of electron transport during ischemia with amobarbital, an inhibitor at the rotenone site of complex I, protects mitochondria against ischemic damage. Amobarbital treatment immediately before ischemia was used to test the hypothesis that damage to mitochondrial respiration occurs mainly during ischemia and that protection of mitochondria during ischemia leads to decreased cardiac injury with reperfusion. Langendorff-perfused Fischer-344 rat hearts were treated with amobarbital (2.5 mM) or vehicle for 1 min immediately before 25 min of global ischemia. Both groups were reperfused for 30 min without additional treatment. Subsarcolemmal (SSM) and interfibrillar (IFM) populations of mitochondria were isolated after reperfusion. Ischemia and reperfusion decreased state 3 and increased state 4 respiration rate in both SSM and IFM. Amobarbital treatment protected oxidative phosphorylation measured following reperfusion and improved the coupling of respiration. Cytochrome c content measured in SSM and IFM following reperfusion decreased in untreated, but not in amobarbital-treated, hearts. H(2)O(2) release from SSM and IFM isolated from amobarbital-treated hearts during reperfusion was markedly decreased. Amobarbital treatment before ischemia improved recovery of contractile function (percentage of preischemic developed pressure: untreated 51 +/- 4%, n = 12; amobarbital 70 +/- 4%, n = 11, p < 0.01) and substantially reduced infarct size (untreated 32 +/- 2%, n = 7; amobarbital 13 +/- 2%, n = 7, p < 0.01). Thus, mitochondrial damage occurs mainly during ischemia rather than during reperfusion. Reperfusion in the setting of preserved mitochondrial respiratory function attenuates the mitochondrial release of reactive oxygen species, enhances contractile recovery, and decreases myocardial infarct size.

Amobarbital↗
PubMed2006

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.

Aging↗
PubMed2006

Oxidative phosphorylation and aging.

This review addresses the data that support the presence and contribution of decreased mitochondrial oxidative phosphorylation during aging to impaired cellular metabolism. Aging impairs substrate oxidation, decreases cellular energy production and increases the production of reactive intermediates that are toxic to the cell. First, the basic principles of mitochondrial oxidative physiology are briefly reviewed. Second, the focus on the relationship of altered mitochondrial respiration to the increased production of reactive oxygen species that are employed by the "rate of living" and the "uncoupling to survive" theories of aging are discussed. Third, the impairment of function of respiration in aging is reviewed using an organ-based approach in mammalian systems. Fourth, the current state of knowledge regarding aging-induced alterations in the composition and function of key mitochondrial constituents is addressed. Model organisms, including C. elegans and D. melanogaster are included where pertinent. Fifth, these defects are related to knowledge regarding the production of reactive oxygen species from specific sites of the electron transport chain.

Aging↗
PubMed2006

Reversal of mitochondrial defects before ischemia protects the aged heart.

Myocardial injury is increased in the aged heart during ischemia and reperfusion. Aging decreases oxidative metabolism in interfibrillar mitochondria (IFM) located between the myofibrils. We asked whether reversal of aging defects in IFM before ischemia would decrease injury in the aged heart following ischemia and reperfusion. Treatment with acetylcarnitine (AcCN) increases the activity of cytochrome oxidase in the aged heart. Aged (24 months) and adult (6 months) Fischer 344 rats were treated with AcCN (300 mg/kg i.p. 3 h before excision of the heart) or served as controls. AcCN restored oxidative phosphorylation and the activity of complexes III and IV in IFM from aged hearts to rates present in adults. Isolated hearts underwent 25 min global ischemia and 30 min reperfusion without additional treatment. Contractile recovery during reperfusion improved in hearts from AcCN-treated aged rats compared to aged controls and were similar to adults in recovery. AcCN-treated aged hearts sustained less damage, indicated by decreased lactate dehydrogenase (LDH) release during reperfusion. AcCN treatment did not alter functional recovery or LDH release in adults. Restoration of mitochondrial function in the aged heart before ischemia was accompanied by enhanced contractile recovery and decreased tissue injury following ischemia and reperfusion.

Acetylcarnitine↗
PubMed2005

Depletion of cardiolipin and cytochrome c during ischemia increases hydrogen peroxide production from the electron transport chain.

Mitochondrial electron transport is a major source of reactive oxygen species (ROS) during cardiac ischemia and reperfusion. In the isolated rabbit heart, 30 and 45 min of ischemia decrease the contents of cardiolipin and cytochrome c in subsarcolemmal mitochondria (SSM) located beneath the plasma membrane. In contrast, interfibrillar mitochondria (IFM) in the interior of the myocyte do not sustain a decrease in cardiolipin. We proposed that the depletion of cardiolipin and the accompanying cytochrome c loss during ischemia were critical events that amplified ROS production by mitochondria. The total production of H2O2 was measured in submitochondrial particles (SMP) prepared from rabbit heart SSM and IFM after 0, 15, 30, and 45 min of ischemia. With NADH as substrate, total H2O2 production was increased only in SMP from SSM after 30 and 45 min ischemia, when ischemia decreased the content of cardiolipin and cytochrome c. In contrast, ischemia did not augment H2O2 generation in SMP from IFM with preserved cardiolipin and cytochrome c content. Thus, during the evolution of ischemic injury, H2O2 production from the electron transport chain increased after depletion of cardiolipin and the loss of cytochrome c.

Animals↗
PubMed2005

Blockade of electron transport before cardiac ischemia with the reversible inhibitor amobarbital protects rat heart mitochondria.

Cardiac ischemia damages the mitochondrial electron transport chain. Irreversible blockade of electron transport at complex I by rotenone decreases ischemic damage to cardiac mitochondria by decreasing the loss of cytochrome c and preserving respiration through cytochrome oxidase. Therapeutic intervention to protect myocardium during ischemia and reperfusion requires the use of a reversible inhibitor that allows resumption of oxidative metabolism during reperfusion. Amobarbital is a reversible inhibitor at the rotenone site of complex I. We asked whether amobarbital administered immediately before ischemia protected respiratory function. Isolated rat hearts were perfused for 15 min followed by 25-min global ischemia at 37 degrees C. Amobarbital-treated hearts received drug for 1 min before ischemia. Subsarcolemmal (SSM) and interfibrillar (IFM) populations of mitochondria were isolated after ischemia, and oxidative phosphorylation was measured. Amobarbital protected oxidative phosphorylation, including through cytochrome oxidase, in both SSM and IFM in a dose-dependent manner, with an optimal dose of 2 to 2.5 mM. Amobarbital also preserved cytochrome c content in both SSM and IFM. Thus, reversible blockade of the electron transport chain during ischemia protects mitochondrial respiration.

Amobarbital↗
PubMed2005

Cardioplegia prevents ischemia-induced transcriptional alterations of cytoprotective genes in rat hearts: a DNA microarray study.

BACKGROUND: Energy conservation and calcium homeostasis contribute to myocardial protection provided by hyperkalemic cardioplegia during ischemia. Complimenting these established mechanisms of protection, previous work suggested that activation of cytoprotective signaling pathways also contributes to reduced injury with cardioplegia. We proposed that cardioplegia would recruit cytoprotective pathways and investigated the transcriptional response of the heart after cardioplegia-protected ischemia compared with that after ischemia alone. METHODS: Isolated perfused rat hearts underwent 40 minutes of global ischemia alone or with St Thomas cardioplegia, followed by 120 minutes of reperfusion. The expression profiles of isolated RNA were determined by using Affymetrix microarrays and assessed by comparing cardioplegia-protected hearts and hearts undergoing unprotected ischemia with time-matched control hearts. The content of selected proteins was assessed by means of immunoblotting. RESULTS: Cardioplegia preserved the expression of multiple genes involved in carbohydrate and fatty acid metabolism, glycolysis, and electron transport compared with ischemia alone. The expression of the sodium-calcium exchanger and ryanodine receptor was preserved in line with the ability of cardioplegia to decrease calcium overload. The expression of multiple cytoprotective molecules, including protein-tyrosine kinase, calcineurin B, p38 mitogen-activated protein kinase, voltage-dependent anion channel, protein kinase C , heat shock protein 70, and manganese superoxide dismutase all showed decreased expression in ischemia but were preserved to near nonischemic levels by cardioplegia. CONCLUSION: Cardioplegia during ischemia maintained an expression profile similar to that seen in nonischemic hearts for genes involved in energy conservation, calcium homeostasis, and cytoprotective pathways, whereas ischemia alone did not. Exposing the transcriptional differences in cytoprotective genes during untreated and cardioplegia-treated ischemia provides valuable insight into an additional mechanism of cardioprotection induced by cardioplegia.

Animals↗
PubMed2004

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.

Animals↗
PubMed2004

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.

Animals↗
PubMed2003

Ischemia-reperfusion injury in the aged heart: role of mitochondria.

The aged heart sustains greater injury during ischemia and reperfusion compared to the adult heart. Aging decreases oxidative phosphorylation and the activity of complexes III and IV only in interfibrillar mitochondria (IFM) that reside among the myofibrils, whereas subsarcolemmal mitochondria (SSM), located beneath the plasma membrane, remain unaltered. The peptide subunit composition of complexes III and IV is intact in aging. The aging defect in complex IV is in the inner membrane lipid environment. The defect in complex III is within the ubiquinol binding site of the cytochrome b subunit. Following ischemia, in the aged heart both SSM and IFM sustain additional decreases in complex III and complex IV activity. In contrast to the aging defect, with ischemia the subunits of complex IV appear to be damaged. Ischemia inactivates the iron-sulfur peptide subunit in complex III. Mitochondria are the major source of the reactive oxygen species that are generated during myocardial ischemia. Complex III is the major site of mitochondrial oxyradical production during ischemia in the adult heart. The role of complex III in the oxidative damage sustained by the aged heart during ischemia, as well as the potential contribution of aging defects in electron transport to ischemic damage in the aged heart, deserves further study. We propose that following ischemic damage to the electron transport chain, the production and release of reactive oxygen species increases from mitochondria in the aged heart, leading to additional damage during reperfusion.

Age Factors↗
PubMed2003

Production of reactive oxygen species by mitochondria: central role of complex III.

The mitochondrial respiratory chain is a major source of reactive oxygen species (ROS) under pathological conditions including myocardial ischemia and reperfusion. Limitation of electron transport by the inhibitor rotenone immediately before ischemia decreases the production of ROS in cardiac myocytes and reduces damage to mitochondria. We asked if ROS generation by intact mitochondria during the oxidation of complex I substrates (glutamate, pyruvate/malate) occurred from complex I or III. ROS production by mitochondria of Sprague-Dawley rat hearts and corresponding submitochondrial particles was studied. ROS were measured as H2O2 using the amplex red assay. In mitochondria oxidizing complex I substrates, rotenone inhibition did not increase H2O2. Oxidation of complex I or II substrates in the presence of antimycin A markedly increased H2O2. Rotenone prevented antimycin A-induced H2O2 production in mitochondria with complex I substrates but not with complex II substrates. Catalase scavenged H2O2. In contrast to intact mitochondria, blockade of complex I with rotenone markedly increased H2O2 production from submitochondrial particles oxidizing the complex I substrate NADH. ROS are produced from complex I by the NADH dehydrogenase located in the matrix side of the inner membrane and are dissipated in mitochondria by matrix antioxidant defense. However, in submitochondrial particles devoid of antioxidant defense ROS from complex I are available for detection. In mitochondria, complex III is the principal site for ROS generation during the oxidation of complex I substrates, and rotenone protects by limiting electron flow into complex III.

Animals↗
PubMed2003

Aging defect at the QO site of complex III augments oxyradical production in rat heart interfibrillar mitochondria.

Complex III in the mitochondrial electron transport chain is a proposed site for the enhanced production of reactive oxygen species that contribute to aging in the heart. We describe a defect in the ubiquinol binding site (Q(O)) within cytochrome b in complex III only in the interfibrillar population of cardiac mitochondria during aging. The defect is manifested as a leak of electrons through myxothiazol blockade to reduce cytochrome b and is observed whether cytochrome b in complex III is reduced from the forward or the reverse direction. The aging defect increases the production of reactive oxygen species from the Q(O) site of complex III in interfibrillar mitochondria. A greater leak of electrons from complex III during the oxidation of ubiquinol is a likely mechanism for the enhanced oxidant production from mitochondria that contributes to aging in the rat heart.

Aging↗
PubMed2003

Cardioprotection by St Thomas' solution is mediated by protein kinase C and tyrosine kinase.

BACKGROUND: Intracellular signaling pathways, specifically the activation of protein kinase C and tyrosine kinase, are essential to the cardioprotection of ischemic preconditioning. We proposed that activation of PKC and TK contribute to the myocardial protection of St. Thomas' No. 2 cardioplegia solution (STC). MATERIALS AND METHODS: Isolated rat hearts were exposed to 40 min of global ischemia followed by 120 min of reperfusion. Before ischemia, hearts received no treatment (control; n = 7), STC (n = 7), phorbol 12-myristate 13-acetate (PMA; n = 6), PMA + chelerythrine (n = 6), anisomycin (n = 6), anisomycin + genistein (n = 7), STC + chelerythrine (n = 7), STC + genistein (n = 7), PMA + genistein (n = 7) or anisomycin + chelerythrine (n = 7). Left ventricular developed pressure (LVDP) recovery, myocardial infarct size, and lactate dehydrogenase release were measured. RESULTS: STC as well as PMA (protein kinase C activator) and anisomycin (tyrosine kinase activator) significantly reduced infarct size (6.9 +/- 2.9%, 9.6 +/- 2.1%, 14.0 +/- 4.4%) compared with controls (42.4 +/- 2.9%, P < 0.05). The infarct reduction of PMA and anisomycin were blocked by their inhibitors chelerythrine and genistein, respectively. Both chelerythrine (29.2 +/- 4.1%, P < 0.05) and genistein (40.4 +/- 4.3%, P < 0.05) attenuated the reduction of infarct size provided by STC. The recovery of LVDP improved with STC, PMA and anisomycin (72.6 +/- 1.4%, 60.4 +/- 4.7%, 57.2 +/- 4.6%) compared with control (33.8 +/- 3.6%, P < 0.05). Addition of chelerythrine or genistein to STC impaired recovery of LVDP (52.3 +/- 4.4%, 35.1 +/- 2.5%, P < 0.05) compared with STC treatment. CONCLUSION: Administration of the pharmacologic inhibitors chelerythrine and genistein blunts the cardioprotection caused by STC treatment.

Animals↗
PubMed2002

Interfibrillar cardiac mitochondrial comples III defects in the aging rat heart.

We used the Fischer 344 rat as a model for aging 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 apoprotein. Thus, at the onset of reperfusion, complex 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.

Aging↗
PubMed2002

Preservation of cardiolipin content during aging in rat heart interfibrillar mitochondria.

Aging selectively decreases the rate of oxidative phosphorylation in the interfibrillar population of cardiac mitochondria (IFM) located between the myofibers. In contrast, subsarcolemmal mitochondria (SSM), located below the plasma membrane, remain unaffected. IFM from elderly (24-month-old) Fischer 344 rats have a decreased specific activity of complexes III and IV. Complexes III and IV require an inner mitochondrial membrane lipid environment enriched in the oxidatively sensitive phospholipid cardiolipin for maximal activity. We asked if aging decreases the content or alters the composition of cardiolipin as a potential mechanism of the aging defect in IFM. The content and composition of mitochondrial phospholipids were measured in SSM and IFM from adult and aging rat hearts. Aging did not alter the content of mitochondrial phospholipids, including cardiolipin, in either population of mitochondria. The composition of cardiolipin based on characterization of both acyl group and the individual molecular species of cardiolipin was also unaltered by aging. Lipid-mediated oxidative modification of complex III subunits was not detected, making cardiolipin-derived oxidative damage to complex III unlikely. Thus, alterations in cardiolipin are not the mechanism for the aging defect in IFM in Fischer 344 rats.

Aging↗