Antioxidant-sensitive shortening of ventricular action potential in hyperthyroid rats is independent of lipid peroxidation.

The effects of substances able to reduce peroxidative processes on thyroid hormone-induced electrophysiological changes in ventricular muscle fibres were examined. For this study, 60 day old euthyroid and hyperthyroid rats were used. One group of hyperthyroid rats was untreated and the others were treated with vitamin E, N-acetylcysteine, and cholesterol, respectively. Hyperthyroidism was elicited by 10 day treatment with daily i.p. injections of triiodothyronine (10 microg/100 g body weight). Vitamin E and N-acetylcysteine were administered for 10 days by daily i.m. injections (20 mg/100 g body weight) and daily i.p. injections (100 mg/100 g body weight), respectively. Cholesterol was administered by cholesterol-supplemented diet (4%) from day 30. Hyperthyroidism induced a decrease in the whole antioxidant capacity and an increase in both lipid peroxidation and susceptibility to oxidative stress. Vitamin E and N-acetylcysteine administration to hyperthyroid rats led to reduction in lipid peroxidation and susceptibility to oxidative stress and to increase in antioxidant level, while the diet addition of cholesterol decreased lipid peroxidation but did not modify the other parameters. The hyperthyroid state was also associated with a decrease in the duration of the ventricular action potential recorded in vitro. The vitamin E and N-acetylcysteine administration attenuated the thyroid hormone-induced changes in action potential duration, which was however, significantly different from that of the euthyroid rats. In contrast, cholesterol supplementation did not modify the electrical activity of hyperthyroid heart. These results demonstrate that the triiodothyronine effects on ventricular electrophysiological properties are mediated, at least in part, through a membrane modification involving a free radical mechanism. Moreover, they indicate that the antioxidant-sensitive shortening of action potential duration induced by thyroid hormone is likely independent of enhanced peroxidative processes in sarcolemmal membrane.

Free radical involvement in doxorubicin-induced electrophysiological alterations in rat papillary muscle fibres.

OBJECTIVE: This work was designed to determine whether the doxorubicin-induced changes in heart electrical activity are due to increased free radical production and membrane oxidative damage. METHODS: Four groups of rats (60 days old) were used. One group was untreated and the others were treated with doxorubicin (DXR), DXR and vitamin E, and DXR and N-acetylcysteine (NAC), respectively. DXR was administered by single i.p. injection (20 mg/kg b.wt.). Vitamin E was administered by ten daily i.m. injections (100 mg/kg), while NAC (100 mg/kg) was injected i.p. 1 h before and 7 h after DXR. The effectiveness of the drug in inducing oxidative stress in different tissues and of the antioxidants in offering protection was established by determining antioxidant capacity, susceptibility to oxidative stress, and lipid peroxidation in heart, liver, and blood. The drug effect on heart electrical activity was determined by measuring the heart rate in vivo and action potential configuration in papillary muscle fibres in vitro. Heart lipid peroxidation and electrical activity were also examined in both vitamin E and NAC-treated rats. RESULTS: DXR treatment decreased antioxidant capacity and increased lipid peroxidation and susceptibility to oxidative stress in heart and blood, but not in liver. DXR administration to rats treated with antioxidants did not produce significant changes in antioxidant capacity and susceptibility to oxidative stress even in heart and blood. Furthermore, lipid peroxidation in heart and liver from DXR- and vitamin E-treated rats, and in liver from DXR- and NAC-treated rats was lower than in untreated controls. DXR treatment also increased the duration of ventricular action potentials in untreated rats, but not in antioxidant-treated rats. The treatment of control animals with the antioxidants affected lipid peroxidation, but not cardiac electrical activity. CONCLUSIONS: The protection offered by antioxidants against electrophysiological alterations indicates a free radical involvement in such alterations. In contrast, although electrical modifications are associated with increased peroxidative processes and both are prevented by the antioxidants, it is not yet clear whether a causative relationship exists between them.

Effect of phenobarbital treatment on characteristics determining susceptibility to oxidants of homogenates, mitochondria and microsomes from rat liver.

This study was designed to investigate the possible oxidative changes associated with alterations in cytochrome P450 levels in rat liver. Accordingly, extent of peroxidative processes, cytochrome and antioxidant content, capacity to face an oxidative stress were determined in liver microsomes, mitochondria, and homogenates from normal and phenobarbital (PB)-treated rats. Liver content of microsomal and mitochondrial proteins was also determined by the values of the activities of marker enzymes (glucose-6-phosphatase and cytochrome oxidase, respectively) in liver homogenate and in two cellular fractions. The increase in the liver content of microsomal and mitochondrial proteins indicated that PB caused proliferation of both smooth endoplasmic reticulum and mitochondrial population. Treatment with PB also gave rise to a general increase in peroxidative reactions (evaluated measuring malondialdehyde and hydroperoxides (HPs)), in the different cell compartments, even though HPs were not found significantly increased in mitochondrial fraction. The increase in peroxidative processes was associated with significant decreases in antioxidant concentration (expressed in terms of equivalent concentration of an antioxidant, such as the desferrioxamine), in all preparations from PB-treated rats. The response to oxidative stress in vitro (evaluated determining the parameters characterizing light emission from preparations stressed with sodium perborate) showed a substantial PB-induced increase in the susceptibility to oxidative challenge only in liver homogenate. The lack of changes in the mitochondrial preparations is likely due to decrease in concentration of both free radical producing species and antioxidants. The lack of changes in microsomal fraction is apparently in contrast with its lower oxidant capacity and higher content of cytochromes which are able to determine sensitivity to pro-oxidants. However, it could be due to the ability of cytochrome P450 to interact with the active oxygen species formed at its active center.

Effect of thyroid state on lipid peroxidation, antioxidant defences, and susceptibility to oxidative stress in rat tissues.

The effects of altered thyroid states on lipid peroxidation, antioxidant capacity, and susceptibility to oxidative stress of rat tissues were examined. Hypothyroidism was induced by administering methimazole in drinking water for 15 days. Hyperthyroidism was elicited by a 10-day treatment of hypothyroid rats with tri-iodothyronine (10 micrograms/100 g body weight). In tissues of hypothyroid rats the lipid peroxidation was not modified, whereas in hyperthyroid rats lipid peroxidation increased in liver and heart but not in skeletal muscle. The glutathione peroxidase activity increased significantly in heart and muscle of hypothyroid rats and in muscle of hyperthyroid rats. The glutathione reductase activity was not modified in tissues of hypothyroid and hyperthyroid rats. In both rat groups the whole antioxidant capacity of tissues decreased, but significantly only in liver and heart. The results obtained studying the response to oxidative stress in vitro indicated that the susceptibility to oxidative challenge was increased in all tissues of hyperthyroid rats and in heart and muscle of hypothyroid animals. These results are explainable in terms of tissue variations in haemoprotein content and/or of antioxidant capacity. Since it has been reported that hypothyroidism offers in vivo protection against free radical damage, we suggest that such an effect could be due to greater effectiveness of cellular defence systems different from antioxidant ones.

Effect of iodothyronines on electrophysiological properties of rat papillary muscle fibres.

We studied the effect of in vivo administration of 3,3',5-triiodo-L-thyronine (L-T3) and other iodothyronines to thyroidectomized rats on the heart rate and electrophysiological properties, recorded in vitro, from papillary muscle fibres. The treatment with a daily substitutive L-T3 dose (3.7 nmoles/100 g body weight) for ten days was associated with a significant increase of heart rate and reduction of the action potential duration (APD) in comparison to that recorded from thyroidectomized rats. Analogous changes were obtained by treatment with equimolar dose of iodothyronines, such as 3,3',5-triiodo-D-thyronine (D-T3), 3,3',5,5'-L-tetraiodothyronine (L-T4), and 3'-isopropyl-3,5-diiodothyronine (3'-Ip-T2), but not by treatment with 3,3',5'-L-triiodothyronine (L-rT3). Unlike the heart rate, APD was found in close correlation with the relative occupancy by iodothyronines of the heart thyroid hormone-specific nuclear sites reported in literature. Such a pattern was little modified when the stimulation rate of the preparations was increased from 1 Hz to 5 Hz, nearing the physiological rates. In fact, there were no significant APD changes for all groups, and only the significance of the APD difference between the T + L-T3 and T + L-rT3 groups was modified. In accordance to the expectations, L-T4 showed appreciable effects on APD at both frequencies tested, despite its small affinity for nuclear receptors. Indeed, it is well-known that a substantial proportion of the T4 effect derives from peripheral conversion to T3. Therefore, our results support the hypothesis that the long-term effects of thyroid hormone on ventricular electrophysiological properties are induced via synthesis of proteins that are part of ionic channels, through a pathway involving hormone interaction with the cell nucleus.

Modifications of antioxidant capacity and heart electrical activity induced by hydroperoxide in normal and vitamin E-fed rats.

Wistar rats, fed control or vitamin E-supplemented diet, were subjected to oxidative stress by a ten day treatment with daily intraperitoneal dose of tert-butyl hydroperoxide (TBHP) (0.1 mumol/100 g body weight). The effectiveness of both diet and hydroperoxide treatment was established by determining the antioxidant capacity of blood, liver, and heart with an enhanced luminescence method. While the diet addition of vitamin E increased the antioxidant capacity of all the tissues in treated and untreated animals, the hydroperoxide treatment failed to decrease liver antioxidant capacity in control diet fed animals. The effect of the reiterated production of free radicals on electrophysiological properties of myocardium was determined by studying the heart rate in vivo and the time course of the surface electrical activity in papillary muscle fibers in vitro. In vivo, a significant tachycardia was found only in TBHP-treated, normal diet fed rats. The duration of action potential, recorded in Krebs' solution at 26 degrees C, was not affected by diet in untreated animals but was modified by hydroperoxide treatment in a diet-dependent way: shortened in normal diet-fed rats and lengthened in vitamin E-supplemented diet-fed rats. On the basis of analogies with the results of electrophysiological recordings on different preparations subjected to oxidative stress in vitro, we suggest that the changes of action potential duration might depend on relative levels of added pro-oxidant and cell antioxidants.

Vitamin E administration attenuates the tri-iodothyronine-induced modification of heart electrical activity in the rat.

This work was designed to determine whether the thyroid-hormone-induced modifications of heart electrical activity are, at least in part, due to increased free radical production. For this study, 60-day-old euthyroid, hyperthyroid and hyperthyroid vitamin-E-treated rats were used. Hyperthyroidism, elicited by a 10 day treatment with tri-iodothyronine, induced an increase in lipid peroxidation without changing the level of antioxidants. Intraperitoneal vitamin administration to hyperthyroid rats led to a reduction in lipid peroxidation and a non-significant increase in antioxidant level. The hyperthyroid state was also associated with an increase in heart rate measured in vivo and a decrease in the duration of the ventricular action potential recorded in vitro. Administration of vitamin E attenuated the thyroid-hormone-induced changes in heart rate and action potential duration, which were, however, significantly different from those of the control euthyroid rats. These results suggest that vitamin E protects hyperthyroid heart against lipid peroxidation by mechanisms that may be independent of the changes in antioxidant systems. Moreover, the reduction in the tri-iodothyronine effects on heart electrophysiological properties indicates that such effects are mediated, at least in part, through a membrane modification, probably related to increased lipid peroxidation, involving a free radical mechanism.

Action potential configuration in heart papillary muscles from female rats in different thyroid states.

We have studied the effects of thyroidectomy and the in vivo administration of different triiodothyronine (T3) doses in thyroidectomized female rats on electrophysiological properties, measured in vitro, of the anterior and posterior papillary muscle fibers from the right ventricle. In each thyroid state, the action potential duration (APD) measured by stimulating at 1 Hz was shorter for the posterior papillary muscle. APD from both preparations was found significantly lengthened in thyroidectomized animals in comparison to euthyroid controls. APD was shortened owing to treatment of thyroidectomized rats with T3 doses up to 10 micrograms/100 g body weight every second day. Treatment with larger doses of T3 tended to restore the values of APD found for ventricular fibres from both controls and thyroidectomized animals treated with substitutive T3 doses (5 micrograms/100 g body weight every second day). As the stimulation rate was increased from 1 to 5 Hz, APD increased in both preparations of all groups. The changes were of different amounts but the APD difference between the rat groups, which were significant at 1 Hz, remained significant at 5 Hz, while the differences between anterior and posterior preparations were cancelled in animals treated with 50 micrograms of T3 and reversed in those treated with 100 micrograms.

Tissue protection against oxidative stress.

We used an enhanced luminescence technique to study the response of rat tissues, such as liver, heart, muscle and blood, to oxidative stress and to determine their antioxidant capacity. As previously found for liver homogenate, the intensity of light emission (E) of tissue homogenates and blood samples, stressed with sodium perborate, is dependent on concentration, and the dose-response curves can be described by the equation E = a.C/exp(b.C). The b value depends on the antioxidant defence capability of the tissues. In fact, it increases when homogenates are supplemented with an antioxidant, and is correlated with tissue antioxidant capacity, evaluated by two previously set up methods both using the same luminescence technique. Our results indicate that the order of antioxidant capacity of the tissues is liver > blood > heart > muscle. The a value depends on the systems catalysing the production of radical species. In fact, it is related to the tissue level of hemoproteins, which are known to act as catalysts in radical production from hydroperoxides. The equation proposed to describe the dose-response relation is simple to handle and permits an immediate connection with the two characteristics of the systems analysed which determine their response to the pro-oxidant treatment. However, the equation which best describes the above relation for all the tissues is the following: E = alpha. C/exp(beta.C delta). The parameter delta assumes values smaller than 1, which seem to depend on relative amounts of tissue hemoproteins and antioxidants. The extension of the analysis to mitochondria shows that they respond to oxidative stress in a way analogous to the tissues, and that the adherence of the dose-response curve to the course predicted from the equation E = a.C/exp(b.C) is again dependent on hemoprotein content.

Effect of cold exposure on electrophysiological properties of rat heart.

Male rats exposed to the cold (4 degrees C) for five or ten days exhibited modifications in their thyroid state, as documented by increases in serum thyroid hormone levels, to which differently graded modifications of heart weight/body weight ratio, heart rate, and resting metabolic rate were associated. The values of the above mentioned thyroid state indicators returned to those of the control when the animals, kept at cold for ten days, were re-exposed to room temperature (24 degrees C) for an additional 10 days. The configuration of action potentials, recorded in vitro at 26 degrees C from fibres of anterior papillary muscles, was different in control rats of different age and was affected by prolonged cold exposure. In fact, the action potential duration (APD) increased after ten days of cold exposure. In the re-exposed group the APD was not different from that of the controls. Such a pattern was not significantly modified when the stimulation frequency increased from 1 Hz to 5 Hz. The above results suggest that in cold exposure, as in experimental hyperthyroidism, thyroid hormone might exert a cardiac chronotropic effect by modifying heart electrophysiological properties. Thus thyroid hormone should play a basic role during the exposure to cold environment, stimulating the body metabolism and increasing heart rate as a response to the requirement for greater tissue perfusion.

Effect of T3 administration on electrophysiological properties of lizard ventricular muscle fibres.

We investigated the effects on the electrophysiological properties of ventricular muscle fibres from lizards kept at 20 degrees C of mild and severe hyperthyroidism. The hyperthyroidism was induced by a 4-day treatment with either 0.025 or 1.0 microgram triiodothyronine g-1 body weight, documented by increased serum levels of thyroid hormone. Triiodothyronine treatment did not modify the duration of the action potential recorded in vitro at 25 degrees C from ventricular muscles stimulated at 1 Hz. Recordings at higher temperatures were associated with a faster repolarization phase and a decrease of of action potential duration in both euthyroid and hyperthyroid animals. However, in lizards treated with 1.0 microgram triiodothyronine . g-1 body weight, the 90% repolarization recovery times at 30 and 35 degrees C (95.6 +/- 14.9 ms and 53.0 +/- 6.0 ms, respectively), were significantly shorter than normal (177.6 +/- 29.2 and 107.2 +/ 18.1 ms, respectively). Action potential duration was also dependent on stimulation frequency of the preparations. Increased frequency led to significant decrease of the duration of action potentials recorded at 25 degrees C. In euthyroid preparations the reductions in 90% repolarization recovery time, owing to increase in stimulation frequency to 2.5 and 5 Hz, were 19.3 +/- 1.7 and 35.6 +/- 2.0 ms, respectively. In hyperthyroid preparations, the reductions in the 90% recovery time due to stimulus frequency increases varied from 35.4 +/- 1.9 and 58.1 +/- 2.1 ms at low hormone doses to 38.9 +/- 2.0 and 58.2 +/- 2.1 ms at high hormone doses. As a result of these differences, the action potential durations recorded from the two hyperthyroid preparations at high stimulation rates were shorter than from euthyroid preparations. The results obtained suggest that lizard cardiac tissue is responsive to hormone action at low environmental temperature, but the effects of such action become evident when the temperature and heart rate increase.

Effect of thyroid state on cardiac electrical activity of the frog Rana esculenta.

The effects of altered thyroid states on the heart rate and ventricular electrophysiological properties of the frog were examined. Hypothyroidism was induced by a 10-day treatment with propylthiouracil and produced decreased serum-free and total triiodothyronine levels below detectable concentrations. Hyperthyroidism, elicited by a 5-day treatment with triiodothyronine, was associated with increased serum thyroid hormone levels. The hypothyroid state was associated with a significantly decreased heart rate measured in vivo and an increased duration of the action potential recorded in vitro from ventricular fibers. Hyperthyroidism was associated with an increased heart rate and a decreased ventricular action potential duration (APD). The dependence of APD on temperature was affected by thyroid status. An increase from 25 to 30 degrees barely shortened the repolarization phase in hyperthyroids, minimally (13.3%) shortened that in euthyroids, and greatly (43.7%) shortened that in hypothyroids; the APD was similar in euthyroid and hypothyroid frogs. The shortening of the repolarization phase, by increased stimulation frequency, was also greater for hypothyroid frogs. In this case, however, the differences in APD among groups remained significant at all the frequencies tested.

Determination by enhanced luminescence technique of liver antioxidant capacity.

A simple approach to quantitative determination of antioxidant capacity of rat liver homogenate is proposed. It consists of measuring chemiluminescence generated by a suitable system "detector" for .OH radicals produced from sodium perborate. The system generating the light signal contained luminol and compounds producing enhancement of light emission, such as sodium benzoate and indophenol. Two different methods, utilizing the same technique of enhanced luminescence, were set up. In a previous work, a parameter b, contained in the equation, which best describes the dependence of the intensity of light emission (E) on liver homogenate concentration (C) (E = a.C/exp(b.C), was found to be related to the level of antioxidants in the homogenate. Therefore, in the first method, the light emission from several dilutions of both liver homogenates, and homogenate and antioxidant mixtures, stressed with sodium perborate, was detected by a luminometer. The best fitting of data to theoretical equation provided b values, which were introduced in a system of equations relating such values to the antioxidant concentration. The solution of above system supplied the antioxidant concentration in the homogenate in terms of the equivalent concentration of the antioxidant used. In the other method, evaluations of the antioxidant capacity of liver homogenates were obtained by the determination of the ability of 10% homogenates to quench the light emission induced by either peroxidase or cytochrome c in comparison to the ability of antioxidant solutions. Both methods are able to evidence the decrease of the antioxidant concentration of liver homogenates after oxidative stress with ter-butylhydroperoxide. The value of both concentration changes and standard errors indicates that the method using a standard curve obtained with peroxidase, such as catalyst of radical reaction, and deferoxamine, such as antioxidant, is to be preferred.

Enhanced luminescence study of liver homogenate response to oxidative stress.

An enhanced luminescence technique was used to monitor the response of liver homogenates stressed with sodium perborate. Rat liver homogenates were subjected to oxidative stress with sodium perborate, and the light signals, generated by a suitable system, containing luminol and compounds producing enhancement of light emission such as sodium benzoate and indophenol, were detected by a luminometer. The intensity of light emission (E) was found dependent on homogenate concentration (C). When C increased, E at first increased as well and, then, decreased rapidly. The graphic expression of this phenomenon resulted as a curve that can be described by the equation: E = a.C/exp(b.C). It is proposed that the a value represents the capacity of the tissue to catalyze the production of .OH radical species. The b value might be related to the capacity of the tissue to scavenge such radicals, since it increases when homogenates are supplemented with antioxidants and decreases when homogenates are treated with prooxidant. The results obtained by supplementing homogenates with iron containing substances, or using model systems, suggest that cell substances catalyzing the luminescent reaction, such as the hemoproteins, are "scavengers" as well as radical producers. The concentration-emission curve obtained with suitable model system is described by the equation: E = a.C/exp(b.Ck). It is suggested that, using the k value, information can be obtained on the relative capacity of hemoproteins and antioxidant systems to interact with .OH radicals.

Ventricular electrophysiological properties in normal and congenitally hypothyroid neonatal rats.

The serum thyroid hormone levels [total (TT3) and free (FT3) triiodothyronine] and the heart rates were determined in neonatal rats of different ages (1-5-10 days). Thyroid hormone levels increase gradually in the first 10 days of age. The heart rate, tested at a body temperature of 37 degrees C, also increases during the same period. As the increase in heart rate in this phase of rat life is not due to the catecholamines, it is suggested that such an increase might depend on the increased thyroid hormone activity. On the other hand in congenitally hypothyroid rats the levels of both hormones and heart rates are lower than in normal animals of the same age. The electrophysiological properties of ventricular muscle fibres include a longer action potential, irrespective of stimulation frequency, in younger, naturally hypothyroid animals. The duration of action potential is greater in the congenitally hypothyroid animals, at all ages. These data demonstrate that, as in young and adult rats, the age-related modifications in heart rate, found in neonatal rats, might be due to thyroid dependent modifications of cardiac electrophysiology.

Electrophysiological properties of the hyperthyroid rat heart.

We have studied the effects of in vivo administration of different T3 doses to thyroidectomized rats on electrophysiological properties, measured in vitro, of papillary muscle fibers. The treatment with increasing T3 doses was associated with a significant reduction of the action potential duration up to a dose as large as 25 micrograms/100 g body weight every second day. The treatment with larger doses of T3 tended to restore the values of the action potential duration present in animals treated with physiological doses (5 micrograms/100 g body weight every second day). Action potential duration is frequency dependent. As the stimulation rate was increased from 1 to 5 Hz, this duration increased in all groups. However the difference between the rat groups remained significant. The cardiac frequency measured in unanaesthetized rats increased as the T3 doses. Furthermore the intrinsic frequency showed a similar increase, indicating a direct effect of T3 on the pacemaker cells in all thyroid states. The mechanism of this action of the thyroid hormone is not, however clear.

Electrophysiological properties of papillary muscle fibres from euthyroid and hypothyroid chick.

1. PTU treatment-induced hypothyroidism is associated with a significant decrease in the chick heart rate. 2. Hypothyroidism produces a slow onset of bradycardia, indicating a late effect of thyroid hormone decrease. 3. After 15 days treatment, an increase of action potential duration, similar to that reported for several mammal species, has been found. 4. Action potential duration is frequency and temperature dependent, but it has been found to be significantly different in the euthyroid and hypothyroid chicks.

Estimation of parameters of allometric equations.

Accurate parameter estimation of allometric equations is a question of considerable interest. Various techniques that address this problem exist. In this paper it is assumed that the measured values are normally distributed and a maximum likelihood estimation approach is used. The computations involved in this procedure are reducible to relatively simple forms, and an efficient numerical algorithm is used. A listing of the computer program is included as an appendix.

Modification of electrophysiological properties of rat heart with age.

The authors have determined the serum thyroid hormone levels [total (TT3) and free (FT3) triiodothyronine], the heart weight/body weight ratio and the heart rate of differently aged male rats. The variations of these parameters show a modification of thyroid state as a function of ageing. The authors have also recorded, at about 26 degrees C, resting and action potentials from single cells of papillary muscles isolated from the same groups of rats. The animals in the higher thyroid state exhibited a repolarization speed higher than the other animals. The thyroidectomy, performed on 50 day old rats, and T3 treatment of the thyroidectomized rats give rise to modifications of repolarization speed and then of action potential duration analogous to ones obtained in previous study for animals thyroidectomized at 30 days of age. These data demonstrate that the modifications of heart electrophysiological properties with age, are due fundamentally to thyroid state modifications. The results suggest also that the cardiac chronotropism modifications which the rat undergoes as a function of ageing are due to the changes of levels of thyroid hormone which might exert its effect by modifying the ion channel kinetics as well as the cardiac receptors.