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Biomedical subjects

F Di Lisa

Publications and source records attributed to F Di Lisa.

At least 37 records · Page 2Linked to original sources
PubMed1995

Mitochondrial membrane potential in single living adult rat cardiac myocytes exposed to anoxia or metabolic inhibition.

1. The relation between mitochondrial membrane potential (delta psi m) and cell function was investigated in single adult rat cardiac myocytes during anoxia and reoxygenation. delta psi m was studied by loading myocytes with JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'- tetra-ethylbenzimidazolylcarbocyanine iodide), a fluorescent probe characterized by two emission peaks (539 and 597 nm with excitation at 490 nm) corresponding to monomer and aggregate forms of the dye. 2. De-energizing conditions applied to mitochondria, cell suspensions or single cells decreased the aggregate emission and increased the monomer emission. This latter result cannot be explained by changes of JC-1 concentration in the aqueous mitochondrial matrix phase indicating that hydrophobic interaction of the probe with membranes has to be taken into account to explain JC-1 fluorescence properties in isolated mitochondria or intact cells. 3. A different sensitivity of the two JC-1 forms to delta psi m changes was shown in isolated mitochondria by the effects of ADP and FCCP and the calibration with K+ diffusion potentials. The monomer emission was responsive to values of delta psi m below 140 mV, which hardly modified the aggregate emission. Thus JC-1 represents a unique double sensor which can provide semi-quantitative information in both low and high potential ranges. 4. At the onset of glucose-free anoxia the epifluorescence of individual myocytes studied in the single excitation (490 nm)-double emission (530 and 590 nm) mode showed a gradual decline of the aggregate emission, which reached a plateau while electrically stimulated (0.2 Hz) contraction was still retained. The subsequent failure of contraction was followed by the rise of the emission at 530 nm, corresponding to the monomer form of the dye, concomitantly with the development of rigor contracture. 5. The onset of the rigor was preceded by the increase in intracellular Mg2+ concentration ([Mg2+]i) monitored by mag-indo-1 epifluorescence. Since under these experimental conditions intracellular [Ca2+] and pH are fairly stable, the increase in [Mg2+]i was likely to be produced by a decrease in ATP content. 6. The inhibition of mitochondrial ATPase induced by oligomycin during anoxia was associated with a rapid and simultaneous change of both the components of JC-1 fluorescence, suggesting that delta psi m, instead of producing ATP, is generated by glycolytic ATP during anoxia. 7. The readmission of oxygen induced a rapid decrease of the monomer emission and a slower increase of the aggregate emission. These fluorescence changes were not necessarily associated with the recovery of mechanical function.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate↗
PubMed1995

Specific degradation of troponin T and I by mu-calpain and its modulation by substrate phosphorylation.

The degradation of troponin (Tn) subunits by calpain was studied by incubating either isolated cardiac Tns or myocardial cryosections with two different calpain isoenzymes isolated from rat skeletal muscle. Western-blot analysis with monoclonal antibodies against TnI and TnT showed that mu-calpain was at least ten times more active than m-calpain in degrading TnI and TnT both in vitro and in situ. TnC was completely resistant to both proteinase forms. Phosphorylation by cyclic AMP-dependent protein kinase (PKA) isolated from rat skeletal muscle reduced the sensitivity of TnI to degradation. This effect in combination with an increased efficiency of the endogenous inhibitor [Salamino, De Tullio, Michetti, Mengotti, Melloni and Pontremoli (1994) Biochem. Biophys. Res. Commun. 199, 1326-1332] probably reduces the proteolytic activity of calpain in cells on PKA stimulation. Conversely, phosphorylation by protein kinase C (PKC) resulted in a twofold increase in the degradation of TnI. Degradation by m-calpain was not modified by Tn phosphorylation. The different sensitivity to mu-calpain might be related to changes in TnI oligomeric structure. Indeed, on PKC phosphorylation, the apparent molecular mass of TnI calculated from the distribution coefficient of Tn complex in Sephadex G-100 matrix was reduced from 90 to 30 kDa suggesting dissociation of the Tn complex.

Animals↗
PubMed1995

Transglutaminase-catalyzed polymerization of troponin in vitro.

In the presence of calcium ions, tissue transglutaminase catalyzes the polymerization of skeletal muscle troponin to high molecular weight insoluble aggregate. The specific action of transglutaminase is proved by the isolation of glutamyl-spermidine isopeptide derivatives. The process involves mainly the troponin T subunit (TnT), with formation of dimers and trimers of TnT, which were reactive with specific antibodies by immunoblotting. Furthermore when incubation is carried out in the presence of radioactive polyamines, the label is incorporated selectively into TnT subunits.

Animals↗
PubMed1994

Regulation of intracellular free Mg2+ and contraction in single adult mammalian cardiac myocytes.

Studies in isolated cardiac myocytes have increased our understanding of intracellular Ca2+ regulation. Because less is known about Mg2+ regulation, adult rat ventricular myocytes were loaded with the Mg(2+)-sensitive fluorescent probe mag-indo 1, and changes in intracellular Mg2+ concentration ([Mg2+]i) and cell length were examined under a variety of conditions. The fluorescent signal was calibrated intracellularly and found to differ slightly from that for the probe in solution. Roughly 40% of the signal was intramitochondrial; the remainder was localized in the cytosol. Basal [Mg2+]i averaged 1.02 +/- 0.03 mM (n = 53 cells). No change in [Mg2+]i was observed during a single electrically stimulated contraction, and only a minor increase was seen during rapid electrical stimulation, which was expected to raise intracellular Ca2+ concentration ([Ca2+]i) to approximately 1 microM. An acid shift in intracellular pH of approximately 1 pH unit was accompanied by a small change in [Mg2+]i (0.34 +/- 0.03 mM, n = 6, P < 0.05). No change in [Mg2+]i was observed when cells were superfused with 15 mM Mg2+, despite marked changes in contraction. [Mg2+]i more than doubled when cells were depleted of ATP by exposure to hypoxia or metabolic inhibitors. The increase in [Mg2+]i was abrupt and occurred at the time of the failure of contraction, plateauing as rigor contracture developed. Reoxygenation was accompanied by a gradual fall in [Mg2+]i in cells that recovered mechanical function, and in a subset of cells that underwent hypercontracture. Studies in cell suspensions confirmed that rapid cellular energy depletion was accompanied by increases in [Mg2+]i and parallel decreases in ATP. Thus [Mg2+]i was largely insensitive to changes in [Ca2+]i or pHi and extracellular [Mg2+] but was rapidly altered by changes in energy state in a manner that was related to specific changes in cell morphology and contractile function.

Adenosine Triphosphate↗
PubMed1994

Contrasting effects of propionate and propionyl-L-carnitine on energy-linked processes in ischemic hearts.

Propionyl-L-carnitine, unlike L-carnitine, is known to improve myocardial function and metabolism altered during the course of ischemia-reperfusion. In this study, the effect of propionyl-L-carnitine has been compared with that of propionate and carnitine on the performance of rat hearts perfused with a glucose-containing medium either under normoxia, ischemia, or postischemic reperfusion. In the postischemic phase, contractile parameters were partially restored both in the control and in the propionate plus carnitine-treated hearts, were markedly impaired by propionate, and were fully recovered by propionyl-L-carnitine. In addition, propionyl-L-carnitine, but not propionate, reduced the functional decay of mitochondria prepared from the ischemic hearts. Even in normoxic conditions propionate, unlike propionyl-L-carnitine, caused a drastic reduction of free CoA and L-carnitine. The concomitant increase in lactate production and decrease in ATP content might be explained by the inhibition of pyruvate dehydrogenase caused by the accumulation of propionyl-CoA. Indeed, when pyruvate was the only oxidizable substrate, propionate induced a gradual decrease in developed pressure, which was largely prevented by L-carnitine. The protective effect of propionyl-L-carnitine may be a consequence of the anaplerotic utilization of propionate in the presence of an optimal amount of ATP and free L-carnitine.

Animals↗
PubMed1994

Involvement of erythrocyte calpain in glycine- and carnitine-treated isovaleric acidemia.

When a 12-y-old girl suffering from isovaleric acidemia was treated with L-carnitine, there was a considerable increase in her blood and urine concentration of isovalerylcarnitine. When later the patient received an infusion of glycine in place of carnitine, isovalerylcarnitine reverted toward the low levels found in a normal subject. At the end of either treatment, erythrocyte calpain was measured and found to be decreased after carnitine therapy (140 versus 96 U/mg Hb with glycine or carnitine, respectively). Because we have previously shown that the activity of calpain isolated from erythrocytes was markedly modified by isovalerylcarnitine, the present results might be seen as the consequence of the chronic exposure of the patient's red blood cells to high levels of isovalerylcarnitine. The lowered calpain activity was also proved by an increase in erythrocyte band 3 phosphorylation together with an increased erythrocyte fragility after calcium loading in the presence of the ionophore A-23187. Calpastatin, the natural inhibitor of calpain, was only slightly modified.

Calpain↗
PubMed1993

Intramitochondrial free calcium in cardiac myocytes in relation to dehydrogenase activation.

OBJECTIVE: The aim was to quantitate intramitochondrial free Ca2+ ([Ca2+]m) in cardiac myocytes under conditions of stimulation previously shown to cause activation of pyruvate dehydrogenase. METHODS: [Ca2+]m was monitored in single, isolated rat cardiac myocytes using fluorescence microscopy following the loading of the cells with the fluorescent chelating agent indo-1, in its permeant acetoxymethylester form, and the selective quenching of cytosolic fluorescence with MnCl2. The extent of contraction upon electrical stimulation was also measured. RESULTS: Electrical stimulation at 2 Hz and higher frequency raised [Ca2+]m significantly, and this was potentiated by exposure to isoprenaline. However, isoprenaline had no effect in quiescent cells, in which [Ca2+]m was raised above resting values by partial replacement of Na+ in the medium. The mitochondrial uncoupling agent carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) raised [Ca2+]m in unstimulated cells, but lowered it in cells subjected to electrical stimulation at 2 Hz or more, to partial Na+ replacement, or to the alkaloid veratridine. CONCLUSIONS: The values of [Ca2+]m in quiescent myocytes (approximately 100 nmol.litre-1) would be associated with very little activation by Ca2+ of pyruvate dehydrogenase phosphatase, based on determination of K0.5 value of 650 nmol.litre-1 in work with mitochondrial suspensions. By contrast, the values of [Ca2+]m associated with electrical stimulation at 2 Hz or greater in the presence of beta adrenergic activation (> 500 nmol.litre-1) would be associated with significant dehydrogenase activation. The effect of beta adrenergic activation is only seen in the presence of electrical stimulation and probably involves enhancement of systolic transients in cytosol [Ca2+]. Effects of uncoupling agents validate the conclusions on the direction and magnitude of the mitochondrial Ca2+ gradient in situ in living myocytes.

Animals↗
PubMed1993

Altered pyruvate dehydrogenase control and mitochondrial free Ca2+ in hearts of cardiomyopathic hamsters.

The fraction of total pyruvate dehydrogenase in the active, dephosphorylated form is much lower in the glucose-perfused isolated hearts of two myopathic strains of Syrian hamster (BIO 14.6 and TO-2) than in the hearts of healthy control animals (F1B). The myopathic hearts also develop significantly less pressure under these conditions. Experiments with isolated myocytes from the BIO 14.6 heart reveal that intramitochondrial free Ca2+ ([Ca2+]m), a positive effector of pyruvate dehydrogenase interconversion, rises much less in response to a protocol of increased frequency of electrical stimulation and adrenergic stimulation than does [Ca2+]m in cells from the healthy control animals (viz from 248 +/- 15 to 348 +/- 44 nM in BIO 14.6 vs. from 241 +/- 35 to 830 +/- 124 nM in F1B, at 4 Hz). As the concentration of Ca2+ that produces half-maximal activation of pyruvate dehydrogenase within mitochondria is 650 nM, this difference between strains is likely the mechanism of the altered enzyme interconversion. The lesser response of [Ca2+]m to electrical stimulation in the BIO 14.6 cells probably results mainly from smaller systolic transients in cytosolic free Ca2+ in response to excitation of single myocytes from the BIO 14.6 animal. Lowered values of [Ca2+]m within the range described would compromise not only pyruvate dehydrogenase activity, but also flux through the tricarboxylate cycle in the myopathic heart, owing to the sensitivity of 2-oxoglutarate dehydrogenase to Ca2+. This may explain the decreased activity of oxidative phosphorylation and performance of work in the myopathic heart.

Animals↗
PubMed1992

Prolonged propionyl-L-carnitine pre-treatment of rabbit: biochemical, hemodynamic and electrophysiological effects on myocardium.

Recently it has been reported that prolonged treatment with propionyl-L-carnitine, a carnitine derivative, results in a positive inotropic effect. To gain further insight into its mode of action, we pre-treated 253 rabbits for up to 10 days with daily doses of 1 mmol/kg propionyl-L-carnitine or L-carnitine intraperitoneally, using saline-treated animals as control. Twenty-four hours after the last injection, we isolated papillary muscles for electrophysiological investigations. Whole hearts were used in perfusion experiments for biochemical and hemodynamic measurements. In addition, mitochondria were harvested from these hearts for the analysis of their function. Plasma and cardiac levels of free carnitine, along with plasma short-chain acylcarnitines, increased at least two-fold after treatment with carnitine or its propionyl-ester, with concomitant rises in tissue long-chain acylcarnitine and long-chain acyl-CoA. At the time of animal sacrifice, treatment did not increase plasma or tissue propionyl-L-carnitine content. The studies carried out with perfused hearts and isolated mitochondria failed to show an effect of propionyl-L-carnitine pre-treatment on high-energy phosphate metabolism or respiration. Papillary muscles from animals, treated for 10 days, showed a lengthening of the action potential duration from 63 +/- 4 to 102 +/- 6 ms (P less than 0.001) at -10 mV. Perfused hearts from these rabbits displayed positive inotropy, as indicated by an improved pressure development at higher ventricular filling volumes, e.g., from 39 +/- 4 to 60 +/- 3 mmHg (P less than 0.05) at 3.6 ml. Pre-treatment with L-carnitine or saline failed to affect the electrophysiological and hemodynamic variables. Thus, prolonged treatment of rabbits with propionyl-L-carnitine, but not with L-carnitine, improved contractility and lengthened action potential duration in isolated muscle preparations.

Action Potentials↗
PubMed1991

Propionyl-L-carnitine: biochemical significance and possible role in cardiac metabolism.

Propionyl-CoA is formed principally during amino acid catabolism. It is then converted chiefly to succinate in a described three-step sequence. Free propionate is formed from propionyl-CoA to a very limited extent, but this anion can participate in a futile cycle of activation and hydrolysis, which can significantly deplete mitochondrial ATP. Free CoA and propionyl-CoA cannot enter or leave mitochondria, but propionyl groups are transferred between separate CoA pools by prior conversion to propionyl-L-carnitine. This reaction requires carnitine and carnitine acetyl transferase, an enzyme abundant in heart tissue. Propionyl-L-carnitine traverses both mitochondrial and cell membranes. Within the cell, this mobility helps to maintain the mitochondrial acyl-CoA/CoA ratio. When this ratio is increased, as in carnitine deficiency states, deleterious consequences ensue, which include deficient metabolism of fatty acids and urea synthesis. From outside the cell (in blood plasma), propionyl-L-carnitine can either be excreted in the urine or redistributed by entering other tissues. This process apparently occurs-without prior hydrolysis and reformation. It is suggested that heart tissue utilizes such exogenous propionyl-L-carnitine to stimulate the tricarboxylic acid cycle (via succinate synthesis) and that this may explain its known protective effect against ischemia.

Acyl Coenzyme A↗
PubMed1990

Metabolic changes induced by maximal exercise in human subjects following L-carnitine administration.

In double-blind cross-over experiments, ten moderately trained male subjects were submitted to two bouts of maximal cycle ergometer exercise separated by a 3 day interval. Each subject was randomly given either L-carnitine (2 g) or placebo orally 1 h before the beginning of each exercise session. At rest L-carnitine supplementation resulted in an increase of plasma-free carnitine without a change in acid-soluble carnitine esters. Treatment with L-carnitine induced a significant post-exercise decrease of plasma lactate and pyruvate and a concurrent increase of acetylcarnitine. The determination of the individual carnitine esters in urine collected for 24 h after the placebo exercise trial revealed a decrease of acetyl carnitine and a parallel increase of a C4 carnitine ester, probably isobutyrylcarnitine. Conversely, acetylcarnitine was strongly increased and C4 compounds were almost suppressed in the L-carnitine loading trial. These results suggest that L-carnitine administration prior to high-intensity exercise stimulates pyruvate dehydrogenase activity, thus diverting pyruvate from lactate to acetylcarnitine formation.

Acetylcarnitine↗
PubMed1990

Isovalerylcarnitine is a specific activator of the high calcium requiring calpain forms.

Isovalerylcarnitine, a product of the catabolism of L-leucine, is a potent activator of rat calpains isolated from erythrocytes, kidney, liver, skeletal and heart muscle. Only calpains II, but not calpains I, are activated by IVC, with the only exception of rat erythrocyte calpain I, the only species present in these cells which has a Ca2+ requirement higher than that of most calpain I isoenzymes. Activation by IVC involves a dual effect: 1) a ten fold increase in the affinity of calpain for Ca2+, and 2) an increase in the Vmax 1.3-1.6 fold above the values observed with the native enzymes at saturating [Ca2+] as well as with the autolyzed fully active calpain form at 5 microM Ca2+. The increased affinity for calcium results in an increased rate of autoproteolysis of calpain II. Activation by IVC is additive to that promoted by interaction (or association) to phospholipids vesicles. Together these results suggest that IVC may operate as a selective activator of calpain both in the cytosol and at the membrane level; in the latter case in synergism with the activation induced by association of the proteinase to the cell membrane.

Animals↗
PubMed1990

Influence of L-carnitine administration on maximal physical exercise.

The effects of L-carnitine administration on maximal exercise capacity were studied in a double-blind, cross-over trial on ten moderately trained young men. A quantity of 2 g of L-carnitine or a placebo were administered orally in random order to these subjects 1 h before they began exercise on a cycle ergometer. Exercise intensity was increased by 50-W increments every 3 min until they became exhausted. After 72-h recovery, the same exercise regime was repeated but this time the subjects, who had previously received L-carnitine, were now given the placebo and vice versa. The results showed that at the maximal exercise intensity, treatment with L-carnitine significantly increased both maximal oxygen uptake, and power output. Moreover, at similar exercise intensities in the L-carnitine trial oxygen uptake, carbon dioxide production, pulmonary ventilation and plasma lactate were reduced. It is concluded that under these experimental conditions pretreatment with L-carnitine favoured aerobic processes resulting in a more efficient performance. Possible mechanisms producing this effect are discussed.

Administration, Oral↗
PubMed1990

Transport and functions of carnitine in muscles.

The transport, function and metabolism of carnitine are discussed with regard to their importance in clinical chemistry. In humans carnitine is synthesized from protein-derived trimethyllysine in liver, brain and kidney. Muscles take up carnitine from the blood in an exchange-diffusion process with endogenous deoxycarnitine, the immediate precursor of carnitine. Besides catalysing the transport of long-chain acyl groups in mitochondria, carnitine is necessary for the export of intramitochondrially produced short-chain acyl residues and for the trapping and the elimination of unphysiological compounds (benzoic, pivalic, valproic acids etc.). The detection and quantitation in urine of these physiological and unphysiological carnitine esters is necessary for the diagnosis of carnitine deficiencies. The carnitine esters may be eliminated in the urine and/or distributed in tissues, where some of them (acetyl-, propionyl- and isovaleryl-carnitine) may be utilized for specific purposes. The most important carnitine-dependent metabolic disorders are listed according to their causes.

Animals↗
PubMed1990

Effects of temperature on myocardial calcium homeostasis and mitochondrial function during ischemia and reperfusion.

An isolated rabbit heart preparation was used to characterize the effects of hypothermia on the deterioration in mitochondrial respiratory function and on the calcium overload that occurs during ischemia and reperfusion. Hearts were perfused aerobically with an asanguineous solution for 120 minutes or made totally ischemic for 90 minutes at 37 degrees, 34 degrees, 28 degrees, 22 degrees C, respectively, and reperfused for 30 minutes at 37 degrees C. Mitochondrial function was assessed by measuring calcium content, yield, oxygen consumption, and adenosine triphosphate-producing capacities. In addition, the mechanical function of the hearts was measured together with tissue adenosine triphosphate, creatine phosphate, and calcium content. In a separate series of experiments, the effect of temperature on the initial rate of respiration-supported calcium accumulation of mitochondria from freshly excised, nonperfused rabbit hearts was determined. The hearts made ischemic at 37 degrees C were severely depleted of tissue adenosine triphosphate and creatine phosphate. Their mitochondria accumulated calcium and the oxidative phosphorylating activity was impaired. During reperfusion, tissue and mitochondrial calcium levels were substantially increased, state 3 of mitochondrial respiration was further impaired, and the adenosine triphosphate-generating capacities were severely reduced. Diastolic pressure increased and there was no recovery of developed pressure. Isolated mitochondrial function of hearts made ischemic at 28 degrees and 22 degrees C was protected. There was a less marked increase in tissue and mitochondrial calcium, and the initial rate and total production of adenosine triphosphate were maintained. In these hearts there was an almost complete recovery of mechanical performance at reperfusion, whereas the ischemia-induced depletion of tissue adenosine triphosphate and creatine phosphate was not significantly reduced by hypothermia. The hearts made ischemic at 34 degrees C were only partially protected. These data suggest that a decrease in temperature from 37 degrees to 22 degrees C during ischemia did not significantly prevent depletion of adenosine triphosphate at the end of ischemia but reduced tissue and mitochondrial calcium overload, maintaining mitochondrial function. Thus in our experiments the protective effect of hypothermia might be related to a direct reduction of tissue and mitochondrial calcium accumulation rather than to a slowing in rates of energy utilization. This possibility is supported by the finding that in freshly excised, nonperfused rabbit hearts, hypothermia significantly reduced the initial rate of mitochondrial calcium transport.

Animals↗