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Uncoupling protein 1 dependent reactive oxygen species production by thymus mitochondria.
We have previously shown that uncoupling protein 1 is present in thymus and has a role in T-cell development. As reactive oxygen species have been implicated in T-cell development, we set out to determine whether uncoupling protein 1 had the potential to regulate reactive oxygen species production in mitochondria isolated from thymus. This was achieved by inhibiting proton leak through uncoupling protein 1 using the purine nucleotide GDP and through ablation of uncoupling protein 1, measuring the amplex red sensitive reactive oxygen species production by mitochondria. In this work we demonstrate, for the first time, that uncoupling protein 1 has the potential to regulate reactive oxygen species production in thymus mitochondria. We also show that reverse electron transport is possible in thymus mitochondria respiring on succinate and glycerol-3-phosphate. The implications of this regulatory role for uncoupling protein 1 are discussed in the context of thymus function. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.
Insulin, ketone bodies, and mitochondrial energy transduction.
Addition of insulin or a physiological ratio of ketone bodies to buffer with 10 mM glucose increased efficiency (hydraulic work/energy from O2 consumed) of working rat heart by 25%, and the two in combination increased efficiency by 36%. These additions increased the content of acetyl CoA by 9- to 18-fold, increased the contents of metabolites of the first third of the tricarboxylic acid (TCA) cycle 2- to 5-fold, and decreased succinate, oxaloacetate, and aspartate 2- to 3-fold. Succinyl CoA, fumarate, and malate were essentially unchanged. The changes in content of TCA metabolites resulted from a reduction of the free mitochondrial NAD couple by 2- to 10-fold and oxidation of the mitochondrial coenzyme Q couple by 2- to 4-fold. Cytosolic pH, measured using 31P-NMR spectra, was invariant at about 7.0. The total intracellular bicarbonate indicated an increase in mitochondrial pH from 7.1 with glucose to 7.2, 7.5 and 7.4 with insulin, ketones, and the combination, respectively. The decrease in Eh7 of the mitochondrial NAD couple, Eh7NAD+/NADH, from -280 to -300 mV and the increase in Eh7 of the coenzyme Q couple, Eh7Q/QH2, from -4 to +12 mV was equivalent to an increase from -53 kJ to -60 kJ/2 mol e in the reaction catalyzed by the mitochondrial NADH dehydrogenase multienzyme complex (EC 1.6.5.3). The increase in the redox energy of the mitochondrial cofactor couples paralleled the increase in the free energy of cytosolic ATP hydrolysis, delta GATP. The potential of the mitochondrial relative to the cytosolic phases, Emito/cyto, calculated from delta GATP and delta pH on the assumption of a 4 H+ transfer for each ATP synthesized, was -143 mV during perfusion with glucose or glucose plus insulin, and decreased to -120 mV on addition of ketones. Viewed in this light, the moderate ketosis characteristic of prolonged fasting or type II diabetes appears to be an elegant compensation for the defects in mitochondrial energy transduction associated with acute insulin deficiency or mitochondrial senescence.
Lipid accumulation in isolated perfused rat hearts has no apparent effect on mechanical function or energy metabolism as measured by 31P NMR.
Male Sprague-Dawley rats were fed diets that contained 20% by weight soybean oil or rapeseed oil (21% and 43% erucic acid) for 7 days. The rapeseed oil diets increased the cardiac triacylglycerol content 5-fold and 25-fold, respectively, above control values. Hearts were removed from the animals and perfused with modified Krebs-Henseleit buffer at 37 degrees C. The calculated rate-pressure product was used as a measure of contractile function. 31P NMR spectra were acquired throughout a protocol that consisted of 12 min control perfusion, followed by 12 min perfusion with 20 microM isoproterenol, 12 min washout, 12 min total global ischemia, and 28 min reperfusion. The steady state levels of creatine phosphate, ATP, intracellular pH, contractile function, and the free energy of ATP hydrolysis (delta GATP) were determined for all three groups of hearts. Isoproterenol more than doubled the rate-pressure product of the hearts on all diets and decreased the concentrations of creatine phosphate and ATP with a concomitant rise in Pi. After global ischemia, creatine phosphate levels recovered fully, ATP levels remained low, and most hearts developed ventricular fibrillation. Changes in intracellular pH were the same for all groups: pH was 7.1 throughout the equilibration and isoproterenol perfusion period, decreased to pH approximately 6.4 during ischemia, and returned to 7.0 during reperfusion. The results indicate that the fat accumulation that occurs in the hearts of rats fed diets rich in high erucic acid rapeseed oil does not interfere with the cardiac high energy phosphate metabolism or contractile function.(ABSTRACT TRUNCATED AT 250 WORDS)
Analysis of 23Na NMR spectra from isolated perfused hearts.
The 23Na NMR spectra obtained from isolated hearts perfused with buffer containing the paramagnetic shift reagent dysprosium triethylenetetraminehexaacetic acid, Dy(TTHA)3-, are complex and contain a number of overlapping peaks of different intensities. Spectra from rat, rabbit, guinea pig, and ferret hearts obtained during periods of control perfusion are similar and undergo similar changes when the hearts are subjected to periods of ischemia and reflow. The contributions from the intracellular, interstitial, vascular, and bath compartments to the multiple peaks in the spectra of rats hearts have been assigned. The significant contributions to these spectra of bulk magnetic susceptibility effects and incomplete mixing have been demonstrated through a series of modeling experiments. Since the spectra from hearts of different species are so similar, the peak assignments made for the rat are applicable to spectra from rabbit, guinea pig, and ferret hearts as well. This work provides a framework for quantitative analysis of the spectral changes which occur during conditions such as ischemia and reflow.
Energetic correlates of cardiac failure: changes in the creatine kinase system in the failing myocardium.
To address the hypothesis that impaired ATP synthesis rates caused by changes in the creatine kinase system is an important mechanism underlying cardiac failure, we measured total creatine kinase activity, isoenzyme composition and creatine content in two animal models of hypertrophy with cardiac dysfunction, the spontaneously hypertensive rat in the transition to failure and the creatine-depleted hyperthyroid rat heart challenged by hypoxia. During the transition from stable compensated hypertrophy to failure characterized by decreased functional capacity, we found that total creatine kinase activity and particularly mitochondrial creatine kinase activity decreased. The decrease in functional capacity, the further increase in heart size and the derangements in the creatine kinase system did not occur if these animals were treated for 6 months with the antihypertensive agents, guanethidine or hydralazine. These results suggest that changes in the creatine kinase system occur coordinately with the transition to failure. To assess whether the changes in the creatine system may be causally linked to decreased functional capacity, we used 31P NMR spectroscopy of isolated perfused hearts to define the high energy phosphate content and cardiac performance of creatine-depleted (approximately 50%) hypertrophied hearts challenged by hypoxia. These hearts displayed greater susceptibility to hypoxic injury with regard to both systolic and diastolic function during and following hypoxia. We also measured total creatine kinase activity in right ventricular biopsy specimens from patients with various forms of cardiomyopathy and low ejection fractions, and found a positive correlation between total creatine kinase activity and ejection fraction. Taken together, these results support the hypothesis that decreasing the energy reserve for ATP synthesis renders the heart more susceptible to systolic and diastolic failure.
31P NMR spectroscopy of hypertrophied rat heart: effect of graded global ischemia.
To investigate the cause for the greater susceptibility of hypertrophied hearts to ischemic injury, we determined the interrelations of total work output, contractile function and energy metabolism in isolated, perfused normal and hypertrophied rat hearts subjected to graded global ischemia. Cardiac hypertrophy was induced by giving rats seven daily injections of either triiodothyronine (0.2 mg/kg) or isoproterenol (5 mg/kg). All hearts were perfused at an aortic pressure of 100 mmHg in the isovolumic mode in an NMR spectrometer (7.05 Tesla). Heart rate, developed pressure, and coronary flow were monitored simultaneously with changes in pH, creatine phosphate, ATP and inorganic phosphate. During pre-ischemic perfusion, the total work output (rate-pressure product) of hyperthyroid hearts was 28% higher than that of control hearts, whereas hearts from isoproterenol-treated animals showed no difference. However, when related to unit muscle mass, work was normal in hyperthyroid hearts and 26% lower after isoproterenol. Contractile function per unit myocardium (developed pressure/g wet weight) was lower in the hypertrophied hearts. ATP content was the same in all groups. Creatine phosphate decreased 41% after triiodothyronine and 25% after isoproterenol. Inorganic phosphate levels and intracellular pH were similar in control and isoproterenol-treated rat hearts, but were higher in the hyperthyroid rat hearts. The phosphorylation potential and the free energy change of ATP hydrolysis were lowered by hypertrophy, the levels correlating with the depressed contractile function. At each ischemic flow rate, both work and contractile function per unit myocardium were the same for all hearts, but the relations between flow and phosphorylation potential were different for each type of heart. Thus, at low flow rates, hypertrophied hearts perform the same amount of work and have the same contractile function as control hearts, but with abnormal changes in energy metabolism, indicating that the relations of energy status to coronary flow, total work output and contractile function are altered during the process of hypertrophy.
Adenosine production and energy metabolism in ischaemic and metabolically stimulated rat heart.
Adenosine may modulate blood flow and electrical activity in heart in response to changes in myocardial energy metabolism. In the present study, 31P NMR spectroscopy was used to examine the relation between cytosolic phosphate metabolite levels and release of adenosine into the venous effluent of isovolumic heart during graded low-flow ischaemia or metabolic stimulation with isoproterenol. When coronary flow rate was varied in steps between 1.6 and 12 ml/min/g, cytosolic ATP levels did not change significantly but the phosphorylation potential exhibited a linear correlation with flow rate below approximately 7 ml/min/g. Purine release (adenosine and inosine) correlated linearly with the cytosolic phosphorylation potential and free AMP concentration. Metabolic stimulation of hearts with isoproterenol (0.4, 3.0, and 60 nM), produced a significant fall in cytosolic ATP levels and decreased the cytosolic phosphorylation potential. Purine release in these hearts increased exponentially as the cytosolic phosphorylation potential dropped, and as cytosolic free AMP increased. These results support a link between the phosphorylation potential and the mechanism of adenosine production during ischaemia and metabolic stimulation. Presumably, this link is the activity of the enzyme 5'-nucleotidase, which is responsible for converting AMP to adenosine, together with the concentration of its substrate, AMP. In low-flow ischaemia, cytosolic AMP may control adenosine formation. With isoproterenol stimulation, a more complex relationship exists, indicating possible allosteric regulation of the enzyme(s) responsible for adenosine formation, in addition to changes in AMP concentration.
Energy metabolism and contractile function in rat heart during graded, isovolumic perfusion using 31P nuclear magnetic resonance spectroscopy.
The isolated, perfused heart is known to exhibit a linear relation between aortic pressure, coronary flow rate, oxygen consumption and contractile function (rate-pressure product) over a wide range of aortic pressures. Our study sought to determine whether the cytosolic phosphorylation potential [( ATP]/[ADP][Pi]) is the link between mitochondrial respiration and contractile function in this preparation. 31P NMR spectroscopy was used to measure phosphate metabolite levels in isovolumic rat hearts during graded perfusion from 1.6 to 12.8 ml/min/g. It was found that an increase in contractile function paralleled the increase in flow rate, but that marked changes in creatine phosphate, inorganic phosphate and hydrogen ion concentration occurred only at lower flow rates. The cytosolic phosphorylation potential showed a high, positive correlation with contractile function at flow rates below 7.2 ml/min/g, which suggested that mitochondrial respiration was oxygen-limited and that the heart was ischemic. Thus, when oxygen limits myocardial oxidative phosphorylation, cytosolic energy metabolite levels may limit contractile function. At the higher flow rates studied, other metabolic controls may operate to link mitochondrial respiration and workload.
The 31P-nuclear magnetic resonance spectrum of heart: will it be of diagnostic use in clinical cardiology?
Significant progress in two areas will be required before the application of magnetic resonance spectroscopy in clinical cardiology is realized. On the technical side, methods must be developed to allow NMR spectra to be obtained non-invasively from the heart uncontaminated by surrounding tissue and with sufficient spatial resolution and signal to noise to be of practical use. On the application side, the potential value of the information derived from the NMR spectrum to diagnosis must be demonstrated. This review addresses the latter problem by evaluating the information content of the 31P-spectrum of heart within the framework of the practical constraints imposed by its possible clinical application as opposed to its acknowledged research application.
Protein synthesis in the early stages of cardiac hypertrophy.
Cardiac hypertrophy, induced in rats by either tri-iodothyronine or isoproterenol, administered daily for 7 days, was monitored using several parameters. Both treatments increased RNA concentrations 24 hr after the first injection, while heart weight increased following 2 injections to 46% above control after 7 days. Cardiac protein synthetic activity, as determined by the rate of peptidyl-puromycin formation, was increased by both tri-iodothyronine and isoproterenol 24 hr after a single injection, implying an increase in the number of functional ribosomes. RNA activity (the rate of peptidyl-puromycin formation per unit RNA) remained constant, suggesting that neither accelerated rates of initiation or translation nor increased activation of pre-existing, non-translating ribosomes was involved in the observed increase in protein synthetic activity. In contrast, constant infusion of [14C] tyrosine indicated no change in protein synthetic rate 24 hr after a single tri-iodothyronine injection and decreased protein synthetic rate after isoproterenol injection. It is concluded that the use of [3H]puromycin to estimate protein synthetic activity may be a more sensitive procedure for detecting early changes in protein synthesis in cardiac hypertrophy than constant isotope infusion, owing to the problems associated with determining the precise precursor pool for protein synthesis in this latter method.
Osmotic shock: modulation of contractile function, pHi, and ischemic damage in perfused guinea pig heart.
To determine the contribution of changes in extracellular osmolarity to ischemic injury, isolated guinea pig hearts were perfused with hyposmotic (220 mosM) or hyperosmotic (380 mosM) buffer. 31P NMR spectroscopy was used to follow changes in intracellular pH (pHi) and energetics. Hyposmotic buffer decreased myocardial developed pressure by 30 +/- 2% and pHi by 0.02 +/- 0.01 unit, whereas hyperosmotic buffer increased myocardial developed pressure by 34 +/- 1% and pHi by 0.14 +/- 0.01 unit. All hearts recovered to control values on restoration of isosmotic (300 mosM) buffer. The hyperosmolar-induced intracellular alkalosis and developed pressure increase were not prevented by inhibition of Na+/H+ exchange with use of 1 microM HOE-642 but were abolished with use of bicarbonate-free buffers. After 20 min of total global ischemia, hearts perfused with hyposmotic buffer showed significantly greater recoveries of developed pressure, phosphocreatine, and ATP than control hearts, but hearts perfused with hyperosmotic buffer did not recover after ischemia. In conclusion, buffer osmolarities between 220 and 380 mosM alter myocardial pHi and developed pressure but are not deleterious during perfusion. However, buffer osmolarity significantly alters the extent of myocardial ischemic injury.
The beta/alpha peak height ratio of ATP. A measure of free [Mg2+] using 31P NMR.
From 31P NMR measurements made in vitro at 38 degrees C, I = 0.25, pH 5. 75-8.5, and calculated free [Mg2+] from 0 to 5 mM, we show that, within the physiological range of cytosolic free [Mg2+] from 0.25 to 1.5 mM, the chemical shift difference between the alpha- and beta-ATP resonances, deltaalphabeta, changes by only 0.6 ppm. Consequently, we developed new formalisms from known acid and Mg2+ dissociation constants by which the observed chemical shift of Pi, deltaPi, and the peak height ratio of the beta- and alpha-ATP resonances, hbeta/alpha, could be related to free [Mg2+] by simultaneous solution of: [equation: see text] We found that hbeta/alpha changed 2.5-fold as free [Mg2+] varied from 0.25 to 1.5 mM, providing a more sensitive and accurate measure of free cytosolic [Mg2+]. In working rat heart perfused with glucose, free [Mg2+] was 1.0 +/- 0.1 from hbeta/alpha and 1.2 +/- 0.03 from measured [citrate]/[isocitrate] but 0.51 +/- 0.1 from deltaalphabeta. Addition of ketone bodies to the perfusate decreased free [Mg2+] estimated from hbeta/alpha to 0.61 +/- 0.02 and 0.74 +/- 0.11 by [citrate]/[isocitrate] but the estimate from deltaalphabeta was unchanged at 0.46 +/- 0.04 mM. Such differences in estimated free [Mg2+] alter the apparent Keq of the creatine kinase reaction and hence the estimated cytosolic free [SigmaADP].