Loss Of High Energy Phosphates Research Articles

Comprehensive analysis of the cardiac proteome in a rat model of myocardial ischemia-reperfusion using a TMT-based quantitative proteomic strategy

BackgroundTraditional studies of the cardiac proteome have mainly investigated in an animal model by two-dimensional gel electrophoresis (2-DE). However, the results have not been of satisfactory quality for an understanding of the underlying mechanism. Recent quantitative proteomic methods have been improved to overcome these limitations. To comprehensively study the cardiac proteome in a rat model of ischemia-reperfusion (IR), we developed a tandem mass tag (TMT)-based quantitative proteomic strategy. Furthermore, using this strategy, we examined the molecular mechanisms underlying the prevention of myocardial infarction by the intake of Triticum aestivum L. extract (TALE), a representative dietary fiber grain.MethodsCardiac proteomes were analyzed by 2-DE as a gel-based approach, and TMT labeling coupled with two-dimensional liquid chromatography (2D-LC) and tandem mass spectrometry (MS/MS) as a non-gel-based quantitative approach. Additionally, gene ontology annotation was conducted by PANTHER database. Several proteins of interest were verified by a Western blot analysis.ResultsTotal 641 proteins were identified commonly from two independent MS datasets using 2D-LC MS/MS. Among these, we identified 151 IR-related proteins that were differentially expressed between the sham-operation group and IR group, comprising 62 up-regulated proteins and 89 down-regulated proteins. Most of the reduced proteins were involved in metabolic processes. In addition, 57 of the IR-related proteins were affected by TALE intake, representing 25 up-regulated proteins and 32 down-regulated proteins. In particular, TALE intake leads to a switch in metabolism to reduce the loss of high-energy phosphates and the accumulation of harmful catabolites (especially reactive oxygen species (ROS)) and to maintain cytoskeleton balance, leading to a reduction in cardiac IR injury.ConclusionsOur study provides a comprehensive proteome map of IR-related proteins and potential target proteins and identifies mechanisms implicated in the prevention of myocardial infarction by TALE intake in a rat IR model.

Mitochondria, Bioenergetics and Excitotoxicity: New Therapeutic Targets in Perinatal Brain Injury.

Injury to the fragile immature brain is implicated in the manifestation of long-term neurological disorders, including childhood disability such as cerebral palsy, learning disability and behavioral disorders. Advancements in perinatal practice and improved care mean the majority of infants suffering from perinatal brain injury will survive, with many subtle clinical symptoms going undiagnosed until later in life. Hypoxic-ischemia is the dominant cause of perinatal brain injury, and constitutes a significant socioeconomic burden to both developed and developing countries. Therapeutic hypothermia is the sole validated clinical intervention to perinatal asphyxia; however it is not always neuroprotective and its utility is limited to developed countries. There is an urgent need to better understand the molecular pathways underlying hypoxic-ischemic injury to identify new therapeutic targets in such a small but critical therapeutic window. Mitochondria are highly implicated following ischemic injury due to their roles as the powerhouse and main energy generators of the cell, as well as cell death processes. While the link between impaired mitochondrial bioenergetics and secondary energy failure following loss of high-energy phosphates is well established after hypoxia-ischemia (HI), there is emerging evidence that the roles of mitochondria in disease extend far beyond this. Indeed, mitochondrial turnover, including processes such as mitochondrial biogenesis, fusion, fission and mitophagy, affect recovery of neurons after injury and mitochondria are involved in the regulation of the innate immune response to inflammation. This review article will explore these mitochondrial pathways, and finally will summarize past and current efforts in targeting these pathways after hypoxic-ischemic injury, as a means of identifying new avenues for clinical intervention.

Role of mitochondria in apoptotic and necroptotic cell death in the developing brain

Hypoxic–ischemic encephalopathy induces secondary brain injury characterized by delayed energy failure. Currently, therapeutic hypothermia is the sole treatment available after severe intrapartum asphyxia in babies and acts to attenuate secondary loss of high energy phosphates improving both short- and long-term outcome. In order to develop the next generation of neuroprotective therapies, we urgently need to understand the underlying molecular mechanisms leading to cell death. Hypoxia–ischemia creates a toxic intracellular environment including accumulation of reactive oxygen/nitrosative species and intracellular calcium after the insult, inducing mitochondrial impairment. More specifically mitochondrial respiration is suppressed and calcium signaling is dysregulated. At a certain threshold, Bax-dependent mitochondrial permeabilization will occur leading to activation of caspase-dependent and apoptosis-inducing factor-dependent apoptotic cell death. In addition, hypoxia–ischemia induces inflammation, which leads to the release of TNF-α, TRAIL, TWEAK, FasL and Toll-like receptor agonists that will activate death receptors on neurons and oligodendroglia. Death receptors trigger apoptotic death via caspase-8 and necroptotic cell death through formation of the necrosome (composed of RIP1, RIP3 and MLKL), both of which converge at the mitochondria.

High energy phosphates in postischemic myocardium with reduced or normalized function

Mitochondrial damage and decreased levels of high energy phosphates are well-known findings in reversibly injured postischemic myocardium (=stunning). Using an isovolumetrically beating isolated rat heart preparation (Langendorff perfusion apparatus) we investigated the effect of postischemic positive inotropic stimulation on the myocardial high energy phosphate content. During postischemic functional steady state with moderately reduced left ventricular developed pressure (118.7 mmHg after 30 minutes of ischemia vs 136.1 mmHg preischemic), dopamine in a dose to reestablish preischemic function was administered for a 20-minute period. Data were compared with postischemic hearts without positive inotropic stimulation: ATP was 2.5 vs 2.5 (μmol/g ww; phosphocreatine was 5.5 vs 6.7 μmol/g ww. These findings indicate that reversibly injured postischemic myocardium can be stimulated for a prolonged period without loss of high-energy phosphates. Therefore, a failure of the energy-generating apparatus is not the reason for the reversible postischemic dysfunction.

Alpha-Tocopherol Pretreatment Reduces Ischaemia/Reperfusion Injury in Rabbit Skeletal Muscle

Background: Ischaemia/reperfusion (I/R) injury is characterized by the production of oxygen free radicals and the loss of high energy phosphates. α-Tocopherol (vitamin E) has been reported to play a protective role against free radical damage. Therefore, its i. v. supplementation prior to onset of ischaemia was investigated in an established I/R injury model.

Depletion of Energy Metabolites Following Acetylcholinesterase Inhibitor-Induced Status Epilepticus: Protection by Antioxidants

Status epilepticus (SE)-induced neuronal injury may involve excitotoxicity, energy impairment and increased generation of reactive oxygen species (ROS). Potential treatment therefore should consider agents that protect mitochondrial function and ROS scavengers. In the present study, we examined whether the spin trapping agent N- tert-butyl-α-phenylnitrone (PBN) and the antioxidant vitamin E (DL-α-tocopherol) protect levels of high-energy phosphates during SE. In rats, SE was induced by either of two inhibitors of acetylcholinesterase (AChE), the organophosphate diisopropylphosphorofluoridate (DFP, 1.25 mg/kg, sc)- or the carbamate carbofuran (1.25 mg/kg, sc). Rats were sacrificed 1 h or 3 days after onset of seizures by head-focused microwave (power, 10 kW; duration 1.7 s) and levels of the energy-rich phosphates adenosine triphosphate (ATP) and phosphocreatine (PCr) and their metabolites adenosine diphosphate (ADP) and adenosine monophosphate (AMP), and creatine (Cr), respectively, were determined in the cortex, amygdala and hippocampus. Within 1 h of seizure activity, marked declines were seen in ATP (34–60%) and PCr (25–52%). Total adenine nucleotides (TAN=ATP+ADP+AMP) and total creatine compounds (TCC=PCr+Cr) were also reduced (TAN 38–60% and TCC 25–47%). No changes in ATP/AMP ratio were seen. Three days after the onset of seizures, recovery of ATP and PCr was significant in the amygdala and hippocampus, but not in the cortex. Pretreatment of rats with PBN (200 mg/kg, ip, in a single dose), 30 min before DFP or carbofuran administration, prevented induced seizures and partially prevented depletion of high-energy phosphates. Pretreatment with the natural antioxidant vitamin E (100 mg/kg, ip/day for 3 days), partially prevented loss of high energy phosphates without affecting seizures. In controls, citrulline, a product of nitric oxide synthesis, was found to be highest in the amygdala, followed by hippocampus, and lowest in the cortex. DFP- or carbofuran-induced seizures caused elevation of citrulline levels seven- to eight-fold in the cortex and three- to four-fold in the amygdala and hippocampus. These results suggest a close relationship between SE, excitotoxicity and energy metabolism. The involvement of oxidative stress is supported by the findings that DFP and carbofuran trigger an excessive nitric oxide (NO) production in the seizure relevant regions of the brain.

Oxidative stress‐induced metabolic alterations in rat brain astrocytes studied by multinuclear NMR spectroscopy

Oxidative stress in cultured astrocytes exerted by 30-min treatment with 50–200 μM H2O2 caused time- and concentration- dependent effects on cellular metabolism. These changes were accompanied by alterations in cellular morphology. Using 31P nuclear magnetic resonance (NMR) spectroscopy, the data demonstrate that the energy status of the cells was greatly affected directly after the stress, as indicated by the loss of high energy phosphates, i.e., phosphocreatine (PCr) and nucleoside triphosphates (NTP). Oxidative stress also involves a dysregulation of the osmotic control in astrocytes, which is accompanied by a dramatic loss of myo-inositol, taurine, and hypotaurine, as monitored by 1H and 13C NMR spectroscopy. While the energy state of the cells was essentially restored during a 7-hr recovery period, the changes in osmolyte concentrations lasted longer and went on throughout the recovery period. Even after 24-hr recovery, organic osmolyte concentrations were still below the control levels. 13C NMR spectra of astrocyte cell extracts also demonstrated an enhanced glucose metabolism via the pentose phosphate pathway (PPP) and a reduced glycolysis. Additionally, the appearance of 13C glutamate points to a distortion of glutamine synthetase (GS), leading to the accumulation of glutamate. Glycolysis as well as GS activity were back to control levels after 7 hr recovery. Thus, in contrast to the energy metabolism, osmoregulatory processes and complex glucose metabolism was impaired not only directly after oxidative stress, but occurred with a later onset during a 2-hr recovery period, and cells only slowly recovered during the next 24 hr. J. Neurosci. Res. 58: 576–585, 1999. © 1999 Wiley-Liss, Inc.

Oxidative stress-induced metabolic alterations in rat brain astrocytes studied by multinuclear NMR spectroscopy

Oxidative stress in cultured astrocytes exerted by 30-min treatment with 50-200 microM H(2)O(2) caused time- and concentration-dependent effects on cellular metabolism. These changes were accompanied by alterations in cellular morphology. Using (31)P nuclear magnetic resonance (NMR) spectroscopy, the data demonstrate that the energy status of the cells was greatly affected directly after the stress, as indicated by the loss of high energy phosphates, i.e., phosphocreatine (PCr) and nucleoside triphosphates (NTP). Oxidative stress also involves a dysregulation of the osmotic control in astrocytes, which is accompanied by a dramatic loss of myo-inositol, taurine, and hypotaurine, as monitored by (1)H and (13)C NMR spectroscopy. While the energy state of the cells was essentially restored during a 7-hr recovery period, the changes in osmolyte concentrations lasted longer and went on throughout the recovery period. Even after 24-hr recovery, organic osmolyte concentrations were still below the control levels. (13)C NMR spectra of astrocyte cell extracts also demonstrated an enhanced glucose metabolism via the pentose phosphate pathway (PPP) and a reduced glycolysis. Additionally, the appearance of (13)C glutamate points to a distortion of glutamine synthetase (GS), leading to the accumulation of glutamate. Glycolysis as well as GS activity were back to control levels after 7 hr recovery. Thus, in contrast to the energy metabolism, osmoregulatory processes and complex glucose metabolism was impaired not only directly after oxidative stress, but occurred with a later onset during a 2-hr recovery period, and cells only slowly recovered during the next 24 hr.

Myocardial oxygenation during high work states in hearts with postinfarction remodeling.

Postinfarction left ventricular remodeling (LVR) is associated with reductions in myocardial high-energy phosphate (HEP) levels, which are more severe in animals that develop overt congestive heart failure (CHF). During high work states, further HEP loss occurs, which suggests demand-induced ischemia. This study tested the hypothesis that inadequate myocyte oxygen availability is the basis for these HEP abnormalities. Myocardial infarction was produced by left circumflex coronary artery ligation in swine. Studies were performed in 20 normal animals, 14 animals with compensated LVR, and 9 animals with CHF. Phosphocreatine (PCr)/ATP was determined with 31P NMR and deoxymyoglobin (Mb-delta) with 1H NMR in myocardium remote from the infarct. Basal PCr/ATP tended to be decreased in postinfarct hearts, and this was significant in animals with CHF. Infusion of dobutamine (20 microg x kg-1 x min-1 IV) caused doubling of the rate-pressure product in both normal and LVR hearts and resulted in comparable significant decreases of PCr/ATP in both groups. This decrease in PCr/ATP was not associated with detectable Mb-delta. In CHF hearts, rate-pressure product increased only 40% in response to dobutamine; this attenuated response also was not associated with detectable Mb-delta. Thus, the decrease of PCr/ATP during dobutamine infusion is not the result of insufficient myocardial oxygen availability. Furthermore, in CHF hearts, the low basal PCr/ATP and the attenuated response to dobutamine occurred in the absence of myocardial hypoxia, indicating that the HEP and contractile abnormalities were not the result of insufficient oxygen availability.

Induction of necrosis but not apoptosis after anoxia and reoxygenation in isolated adult cardiomyocytes of rat.

Apoptosis is one feature of myocardial damage after ischemia-reperfusion, but the causes for its induction are unclear. The present study was undertaken to investigate whether apoptosis in cardiomyocytes is directly initiated by their sub-lethal injury that results from ischemia-reperfusion. Ischemia was simulated on isolated ventricular cardiomyocytes of adult rats by anoxia in a glucose free medium, pH 6.4. Induction of apoptosis was detected by (1) DNA laddering of genomic DNA, (2) TUNEL positive cells (terminal deoxynucleotidyl transferase-mediated-UTP nick end labelling) and (3) annexinV-fluorescein isothiocyanate (annexinV-FITC) binding to cells under exclusion of propidium iodide. Necrotic cells were identified by (1) staining with both annexinV-FITC and propidium iodide, (2) unspecific DNA degradation and (3) enzyme release. Simulated ischemia caused a > 75% loss of high-energy phosphates within 2 h, which was reversible upon reoxygenation at pH 7.4. Even after 18 h of simulated ischemia, creatine phosphate contents recovered to 55.2 +/- 7.3% of control within 1 h. Apoptosis could be induced by UV irradiation (80 J/m2), H2O2 and the NO-donor N2-acetyl-S-nitroso-D,L-penicillinaminamide. In contrast to this, simulated ischemia and reoxygenation could not induce apoptosis in the cells, but with prolonged ischemia more cells became necrotic. After 18 hours of simulated ischemia and 4 h of reoxygenation 41.2 +/- 10.2% myocytes were necrotic (vs. 6.3 +/- 4.4% of control) and only 1.7 +/- 0.5% (vs. 8.7 +/- 4.6% of control) were apoptotic. The percentage of necrotic cells correlated with an increase in lactate dehydrogenase release from 9.9 +/- 0.6% (of total activity) of normoxic controls to 37.9 +/- 5.1% after 18 h of simulated ischemia and 12 h of reoxygenation. Simulated ischemia-reoxygenation causes necrosis of isolated cardiomyocytes but is not sufficient for induction of apoptosis.

The cardiac adrenergic system in ischaemia: differential role of acidosis and energy depletion.

Acute myocardial ischaemia has been shown to modulate the beta-adrenergic system and to activate protein kinase C. The aim of this study was to investigate if two important components of ischaemia, i.e. energy depletion or acidosis, may contribute to these changes. Isolated rat hearts were perfused either with anoxia (in the absence of oxygen) or with cyanide in the absence of glucose as models of energy depletion with a loss of high energy phosphates. Alternatively, isolated hearts were perfused with acidic modified Krebs-Henseleit solution to induce acidosis. Energy depletion induced by cyanide perfusion leads to an increase of beta-adrenergic receptors (81 +/- 7 vs. 50 +/- 3 fmol/mg protein, p < or = 0.05) comparable to the changes observed in ischaemia, yet without any change of total adenylyl cyclase activity or protein kinase C activity. Similar, yet less pronounced changes were induced by anoxic perfusion. Acidic perfusion, in contrast, promotes a translocation of protein kinase C to the plasma membranes, suggesting its rapid activation. Additionally, an increased total forskolin-stimulated activity of adenylyl cyclase (515 +/- 16 vs. 428 +/- 17 pmol/min/mg, p < or = 0.05) was observed. Both were comparable to the sensitization observed in early ischaemia. In acidosis, the density of beta-adrenergic receptors remained unaltered. These data suggest that the regulation of cardiac beta-adrenergic receptors is susceptible to energy depletion, but not to acidosis, whereas the intracellular enzymes both adenylyl cyclase and protein kinase C may be regulated by intracellular acidosis. This is the first differentiation of distinct components of ischaemia modulating the beta-adrenergic signal transduction pathway. Both components may be operative in concert in acute myocardial ischaemia and may contribute to the regulation of these components of signal transduction observed in acute ischaemia.

Altered Contractility of Rabbit Penile Corpus Cavernosum Smooth Muscle by Hypoxia

Purpose To investigate the effects of severe hypoxia on trabecular smooth muscle contractility. Materials and Methods Strips of rabbit corpus cavernosum were mounted in organ chambers to measure isometric tension. In some experiments intracellular free Ca 2+ concentration and tension were measured by the intracellular fluorescent dye FURA-2 and isometric tension recording simultaneously. Results Contractions elicited by norepinephrine, endothelin-1 or potassium were attenuated under hypoxic conditions (pO 2 approximately 10 mm. Hg). Strips contracted with 20 mM. K sup + under normoxic conditions and then exposed to hypoxia consistently lost the potassium-induced tone. The hypoxia-induced relaxation was not affected by the removal of the endothelium, by treatment with the cyclooxygenase blocker, indomethacin, or with the guanylate cyclase blocker, methylene blue. The potassium channel opener, cromakalim, and adenosine relaxed potassium contracted strips; however, the potassium channel blockers glibenclamide, apamin, barium chloride and charybdotoxin or the adenosine receptor antagonist 8-(p-sulfonyl)theophylline were unable to prevent hypoxia-induced relaxation. Tetraethylammonium, a potassium channel blocker, and the depolarizing agents ouabain and high potassium (80 mM.), partially prevented hypoxia-induced relaxation. The calcium ionophore, ionomycin, had no effect on hypoxia-induced relaxations. Hypoxia, within 2 to 6 minutes, caused a large accumulation in intracellular calcium, concomitant with a loss of trabecular smooth muscle tone. Both these effects could be reproduced by inhibiting oxidative phosphorylation with 2,4, dinitrophenol. Reoxygenation resulted in an immediate recovery of both tone and intracellular calcium levels. Tissues under hypoxic conditions for 30 minutes had a 24 percent and 67 percent decrease in the ATP/ADP ratio and in creatine phosphate concentrations, respectively. Conclusion Hypoxia causes a simultaneous increase in intracellular calcium and relaxation which, we propose, is the consequence of inhibition of oxidative phosphorylation with loss of high energy phosphates, necessary for the homeostasis of Ca 2+ and the contractile mechanism of the cavernosal smooth muscle.

High energy phosphates in postischemic myocardium with reduced or normalized function

Mitochondrial damage and decreased levels of high energy phosphates are well-known findings in reversibly injured postischemic myocardium (=stunning). Using an isovolumetrically beating isolated rat heart preparation (Langendorff perfusion apparatus) we investigated the effect of postischemic positive inotropic stimulation on the myocardial high energy phosphate content. During postischemic functional steady state with moderately reduced left ventricular developed pressure (118.7 mmHg after 30 minutes of ischemia vs 136.1 mmHg preischemic), dopamine in a dose to reestablish preischemic function was administered for a 20-minute period. Data were compared with postischemic hearts without positive inotropic stimulation: ATP was 2.5 vs 2.5 (μmol/g ww; phosphocreatine was 5.5 vs 6.7 μmol/g ww. These findings indicate that reversibly injured postischemic myocardium can be stimulated for a prolonged period without loss of high-energy phosphates. Therefore, a failure of the energy-generating apparatus is not the reason for the reversible postischemic dysfunction.

Microvascular incompetence and the failure of hearts to recover contractile function after cardioplegia.

The relationship between the development of microvascular incompetence and the loss of potential for functional recovery following cardioplegia was investigated using St. Thomas' Hospital No. 2 solution (STH) in isolated working rat hearts. Cardiac function was measured prior to cardioplegia and again after 30 min of reperfusion at 37 degrees C following 1, 2 or 4 h arrest at 30 degrees C (n=5). The hearts were then fixed by perfusion with 2.5% glutaraldehyde and then nuclear track emulsion was perfused as an intravascular marker of competent capillaries. Following cardioplegia for 1 h hearts showed 95.4% recovery of aortic flow in the working mode, and a high proportion of the capillaries in the subendocardial (84.6 +/- 2.3%), middle (94.6 +/- 3.0%) and subepicardial (89.1 +/- 4.9%) thirds of the left ventricular myocardium transmitted perfusate. Two hours arrest resulted in significantly diminished recovery of left ventricular function (aortic flow: 56.6 +/- 7.6% and aortic pressure: 64.4 +/- 2.5% and heart rate 56.0 +/- 23.1%). This loss of the remaining two thirds of the potential for functional recovery was associated with significant (P<0.02) reductions in the proportions of competent capillaries (subendocardial, middle and subepicardial thirds to 10.9%, 19.2% and 14.2%, respectively). These non-functional capillaries had open lumina and showed no sign of structural alteration, obstruction or compression, although some focal collections of myocytes (<30%) showed evidence of reperfusion damage including contraction band necrosis. Despite reductions in microvascular competence overall, coronary flow rates (non-working) did not decline, suggesting shunting via large arterio-venous channels. It seems likely that the loss of the first third of the potential for rapid functional recovery following cardioplegia is due to loss of high energy phosphates, whereas the loss of the remaining two-thirds is associated with endothelial cell mediated constriction of small arterial vessels which produces the capillary incompetence demonstrated in this study.

Cell and tissue responses of a murine tumour to phthalocyanine-mediated photodynamic therapy

Mice bearing a subcutaneously growing tumour (Colo 26) were injected intravenously with the photosensitiser chloroaluminium sulphonated phthalocyanine (5 mg/kg) 24 h prior to irradiating the tumour with laser light (675 nm; 50mW, 100 J/tumour). Energy status of the tumour, as assessed by the loss of high energy phosphates in the 31P-nuclear magnetic resonance spectra, was altered dramatically following treatment, such that the ATP fell to undetectable levels within 1 h of light irradiation. However, assessment of the clonogenic capacity of neoplastic cells isolated from dissociated tumours showed that these rapid changes in cellular metabolism were not reflected in similar rapid changes in cell viability. Reductions in clonogenic capacity, which fell to less than 0.1% of control values at 24 h postirradiation, closely mirrored those resulting from the cessation of vascular perfusion. Evaluation of tumour blood flow, using the technique of hydrogen washout, showed that the treatment protocol evoked a gradual and selective reduction in flow within the tumour resulting in complete vascular stasis by approximately 5 h after treatment. The results indicate that while chloroaluminium sulphonated phthalocyanine-mediated photodynamic therapy caused early metabolic damage in neoplastic cells, loss of viability paralleled the induction of complete inhibition of vascular flow in the tumour.

Ischemic preconditioning slows energy metabolism and delays ultrastructural damage during a sustained ischemic episode.

We have shown previously that preconditioning myocardium with four 5-minute episodes of ischemia and reperfusion dramatically limited the size of infarcts caused by a subsequent 40-minute episode of sustained ischemia. The current study was undertaken to assess whether the same preconditioning protocol slowed the loss of high energy phosphates, limited catabolite accumulation, and/or delayed ultrastructural damage during a sustained ischemic episode. Myocardial metabolites and ultrastructure in the severely ischemic subendocardial regions were compared between control and preconditioned canine hearts. Hearts (four to 10 per group) were excised after 0, 5, 10, 20, or 40 minutes of sustained ischemia. All groups had comparable collateral blood flow. Preconditioned hearts developed ultrastructural injury more slowly than controls; evidence of irreversible injury was observed after 20 minutes in controls but not until 40 minutes in preconditioned hearts. Furthermore, after 40 minutes of ischemia, irreversible injury was homogeneous in controls but only focal in preconditioned myocardium. Preconditioning reduced starting levels of ATP by 29%. Nevertheless, it also slowed the rate of ATP depletion during the episode of sustained ischemia, so that after 10 minutes of ischemia, preconditioned hearts had more ATP than controls. However, after 40 minutes, ATP contents were not significantly different between groups. Preservation of ATP resulted from reduced ATP utilization and was not due to increased ATP production. Accumulation of purine nucleosides and bases (products of adenine nucleotide degradation) was limited in preconditioned myocardium. Accumulation of glucose-1-phosphate, glucose-6-phosphate, and lactate also was reduced markedly by preconditioning, due to reduced rates of glycogen breakdown and and anaerobic glycolysis. We propose that preconditioning reduces myocardial energy demand during ischemia, which results in a reduced rate of high energy phosphate utilization and a reduced rate of anaerobic glycolysis. Either preservation of ATP or reduction of the cellular load of catabolites may be responsible for delaying ischemic cell death.

Adenosine attenuates loss of high energy phosphates in ischemic myocardium evaluated by phosphorus NMR spectroscopy

Adenosine attenuates loss of high energy phosphates in ischemic myocardium evaluated by phosphorus NMR spectroscopy

Time course of structure, function, and metabolic changes due to an exogenous source of oxygen metabolites in rat heart

Effects of xanthine (2 mM) and xanthine oxidase (10 U/L) perfusion on myocardial function, lipid peroxide content, high-energy phosphates and their metabolites, and ultrastructure were examined in isolated perfused rat hearts to define the time course of myocardial injury due to exogenous supply of active oxygen species. Peak-developed force and dF/dt showed a decline within 5 min and complete contractile failure was seen at 20 min. Resting tension was higher at 10 min and reached a maximum value of 400% at 40 min. These changes in contractile parameters were reduced by superoxide dismutase (1.2 x 10(5) U/L), catalase (2 and 4 X 10(4) U/L), and mannitol (10 and 20 mM). Lipid peroxide content was significantly higher at 5 min and rose continuously with xanthine-xanthine oxidase (X-XO) perfusion. A close correlation was noted (r = 0.935) between increased lipid peroxide content and a decrease in peak-developed force. Creatine phosphate and adenosine triphosphate (ATP) showed a time-dependent decrease due to X-XO perfusion. Loss of ATP also correlated (r = 0.819) with the contractile failure. Adenosine diphosphate showed an increase at 5 min followed by a decrease at 20 and 40 min. Adenosine monophosphate, adenosine, and creatine content increased with X-XO perfusion. In a semiquantitative morphometric study, significant myocardial and vascular changes became apparent only after 10 min of X-XO perfusion. When a 5-min perfusion with X-XO was followed by a control perfusion, a recovery of developed force and normal structure was noted at 40 min. These data show that X-XO induced contractile failure involves partially reduced forms of oxygen such as superoxide, hydroxyl radicals, and hydrogen peroxide. The negative inotropic effect of a vascular supply of these active oxygen species may be related to increased lipid peroxidation as well as the loss of high-energy phosphates. Structural damage to myocytes and blood vessels and a rise in resting tension were delayed events requiring a continuous and longer exposure to radical species.

Metabolic changes in rabbit spinal cord after trauma: magnetic resonance spectroscopy studies.

Combined phosphorus and proton magnetic resonance spectroscopy (MRS), using double-tuned surface coils, was used to monitor certain metabolic changes in the L-3 spinal segment of anesthetized rabbits prior to and following experimental spinal cord trauma. Following severe trauma, resulting in spastic paraplegia, there was a delayed and progressive accumulation of lactic acid, a decline in intracellular pH, and a loss of high-energy phosphates. Maximal alterations occurred between 2 and 3 hours after the trauma, with little further change by 4 hours. Histological examination 2 weeks after trauma showed tissue necrosis and cavitation. These findings support the concept of secondary tissue injury after spinal cord trauma and suggest that early changes in metabolism, as shown by MRS, may predict irreversible tissue damage.

The pathophysiology of acute myocardial infarction.

Recent work has now clearly established that coronary arterial thrombosis is the direct cause of acute myocardial infarction. This thrombotic event occurs when a pre-existing atherosclerotic plaque ruptures or fissures, thereby exposing underlying thrombogenic material to the circulation. Platelets are thus activated and the clotting cascade is initiated. It is as yet unclear why a previously stable atherosclerotic plaque should fissure or rupture. However, suggested mechanisms include release of vasoactive substances from activated platelets, coronary arterial vasomotion, mechanical stress fatigue of the atherosclerotic plaque, and rupture of vasa vasorum within the atherosclerotic plaque. The resultant cessation of myocardial blood flow produces specific biochemical and physiological alterations secondary to myocardial ischemia. Intracellular acidosis, loss of high-energy phosphates, reduced sensitivity of contractile proteins to calcium, and accumulation of inorganic phosphate and lipid, all occur within the ischemic myocyte. Diastolic compliance is markedly reduced by ischemia followed by cessation of systolic contractile activity. Most of these alterations are reversible if ischemia is relieved promptly. Prolonged ischemia leads to delayed biochemical and physiological recovery and/or cell necrosis.