Magda Horackova

Alterations in electrical and contractile behavior of isolated cardiomyocytes by hydrogen peroxide: Possible ionic mechanisms

The development of H2O2-induced changes in membrane potentials, membrane currents and corresponding contractile activity (shortening) were studied in rat and guinea-pig ventricular myocytes using the suction-pipette whole-cell clamp method. The cells exhibited a different sensitivity to 30 microM H2O2 in terms of time development of the changes, which were fully irreversible. The observed changes are described in three phases: (1) prolongation of action potential duration (APD) accompanied by increased contractility. With a prolonged exposure, the increased APD was accompanied by early afterdepolarizations (EADs), delayed afterdepolarizations (DADs) and aftercontractions. The changes in APD and the EADs were fully and permanently abolished by tetrodotoxin (TTX) but not by nifedipine, while the DADs and aftercontractions were abolished by ryanodine. These changes preceded phase (2), which was characterized by APD shortening, a decrease in contractility, membrane depolarization, single or multiple extrasystoles, or steady spontaneous activity; this phase could not be prevented by any of the above pharmacological interventions and resulted in a final phase (3) characterized by full depolarization and inexcitability. All the above changes were prevented by intracellular application of iron chelator-deferoxamine, indicating that .OH generated intracellularly in the presence of Fe3+ induces the observed changes. The examination of membrane currents indicated that the increased APD may be due to an increase in the TTX-sensitive Na+ current as well as to the decreased delayed current, while L-type Ca2+ channels appear to be unaffected. The shortening of APD during the second phase was associated with a large increase in the delayed K+ current. The increased contractility in the first stage appears to be due to increased sarcolemmal Ca2+ influx via Na(+)-Ca2+ exchange (among other possible mechanisms), leading to a loading of sarcoplasmic reticulum that eventually results in Ca2+ overload and functional failure.

Distribution of intrinsic cardiac neurons in whole-mount guinea pig atria identified by multiple neurochemical coding. A confocal microscope study.

Abstract Functional data indicate that neurons in distinct regions of the heart exert preferential regional cardiac control. To date the regional distribution of specific types of neurons within the intrinsic cardiac nervous system remains unknown, as does their associations with distinct neurotransmitter and/or neuromodulatory profiles. This study was designed to ascertain: (1) the distribution of different classes of neurons within the intrinsic cardiac nervous system as determined by microscopic analysis; (2) the neurochemical profiles of neurons in differing atrial loci; (3) which neurochemicals are co-localized within specific populations of intrinsic cardiac neurons; and (4) the distribution of specific sub-populations of neurons expressing specific immunoreactivities. Taking advantage of confocal laser scanning microscopy and distinct immunoreactive fluorescent markers in various double-label combinations, several sub-populations of intrinsic cardiac neurons were identified. Of all identified neurons, 85–90% were located in ganglia (ganglionic neurons), the rest being isolated (individual neurons). The two general neuronal markers protein gene product 9.5 (PGP 9.5) and microtubule-associated protein (MAP-2) were associated with neurons clustered primarily in the interatrial septum and around the origins of the two vena cavae. Ganglia (group 1) contained three sub-populations of neurons: approx. 80% of ganglionic neurons were large (15–40 µm diameters; group 1a) and approx. 20% had smaller diameters (less than 15 µm; group 1b). All of these neurons were PGP-immunoreactive, exhibiting choline acetyltransferase (ChAT) immunoreactivity (IR), tyrosine hydroxylase (TH) IR, neuropeptide Y (NPY) IR, vasoactive peptide (VIP) IR and substance P (SP) IR. The remaining 5% of ganglionic neurons were small (group 1c; less than 20 µm). These displayed TH immunoreactivity but not MAP, PGP, CHAT, NPY or SP immunoreactivity. Ten to fifteen percent of all neurons loosely distributed outside of ganglia were small (10–25 µm) and located primarily around the origin of the superior vena cava. They displayed immunoreactivity to TH, ChAT, VIP, NPY and SP, but not to MAP-2 or PGP 9.5. These data provide anatomical and immunohistochemical evidence for specific localization of differing populations of intrinsic cardiac neurons with respect to their size, ganglionic distributions and capacity to express multiple neurotransmitters. Although the functional importance of such a regional distribution of differing populations of intrinsic cardiac neurons remains unknown, these anatomical data support the thesis that unique clustering of specific populations of neurons within this nervous system represents the anatomical substrate for complex local cardiac regulatory phenomena occurring at the level of the target organ.

The antioxidant effects of a novel iron chelator salicylaldehyde isonicotinoyl hydrazone in the prevention of H2O2 injury in adult cardiomyocytes

OBJECTIVE This study was designed to investigate the cardioprotective effect of the novel lipophilic iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH) against the oxidative stress exerted by H(2)O(2) through the production of OH radical via the Fenton reaction and to compare them with those of the hydrophilic iron chelator deferoxamine (DFO) and the Na(+)/H(+) exchange inhibitor methylisobutyl amiloride (MIA). METHODS We used long-term cultures of spontaneously beating adult guinea-pig ventricular cardiomyocytes developed and characterized previously in our laboratory. We assessed their contractile activity by video-recording as well as the underlying Ca(i)(2+) transients by Fura 2 fluorescence. In some experiments we also recorded these functional parameters, plus the electrical activity (action potentials) in response to electrical stimulation via suction pipettes, in individual freshly isolated myocytes. RESULTS Exposure of the regularly and synchronously beating cultured cardiomyocytes to 100 microM H(2)O(2) initially caused a substantial prolongation of Ca(i)(2+) transients accompanied by an irregular contractile activity, then in contractile arrest and a severalfold increase in cytosolic [Ca(2+)] that occurred, within 30 min of H(2)O(2) application. Similar effects were also observed using freshly isolated cardiomyocytes. The latter effects were first accompanied by significant prolongation of the action potential duration (APD) with superimposed early afterdepolarizations followed by a second phase with a very fast decrease in APD, contractions, as well as Ca(i)(2+) transients and a third phase of inexcitability, contractile arrest, increased cytoplasmic [Ca(2+)] and a final contracture. All these effects were irreversible in both types of preparations but they could be fully prevented by a 15-min preincubation with 200 microM SIH. Similar protective effects were observed with DFO, but in this case a much higher concentration had to be used (1 mM) and much longer (2 h) preincubation was needed. By contrast, 5 microM MIA failed to fully protect the cardiomyocytes, although a significant delay (10 min) of the effects of H(2)O(2) was observed. CONCLUSIONS The data indicate that SIH provides a very powerful and very fast protection against the oxidative stress exerted by H(2)O(2) presumably via the iron-mediated Fenton reaction producing hydroxyl radical (OH), whereas the protective effect of DFO is hindred by its very slow and rather limited intracellular entry, and the protection that MIA exerts via the inhibition of Na(+)/H(+) exchange against H(2)O(2) much less effective.

Effects of chronic diabetes mellitus on the electrical and contractile activities,45Ca2+ transport, fatty acid profiles and ultrastructure of isolated rat ventricular myocytes

The effects of chronic experimental diabetes on electrophysiological properties, contractile behavior,45Ca2+ transport, fatty acid profiles and ultrastructural characteristics were studied in enzymatically dissociated ventricular myocytes. Diabetes was induced in rats by streptozotocin administration and animals were killed 8–10 weeks later. Myocytes from diabetic rats exhibited electrical behavior similar to that of myocytes from control rats, but their contractile properties were altered. Their sensitivity of the twitch contractions to various positive and negative inotropic agents (isoproterenol, norepinephrine, phenylephrine, acetylcholine, ouabain and veratridine) was greatly diminished. However, a part of the contractile response (the tonic, sustained contractions) were increased in the diabetic myocytes, indicating that the changes are not caused by a decreased sensitivity of myofilaments. Furthermore, the diabetic myocytes exhibited also significant decrease in total Ca2+ content. The fatty acid profile in the diabetic group was changed mainly in that there were slightly elevated levels of docosahexaenoic acid and diminished levels of palmitic acid. The ultrastructure of the diabetic myocytes was affected only slightly. These investigations offer for the first time a comprehensive picture of changes related to diabetic cardiomyopathy as they occur at the level of cardiomyocytes.

Role of peripheral autonomic neurones in maintaining adequate cardiac function

This review has focused on the putative effects that peripheral autonomic neurones exert on cardiac myocytes. Through data obtained by the use of in situ and in vitro models, the unique synaptology and chemical sensitivities of the various types of neurones in intrinsic cardiac and extracardiac intrathoracic ganglia are becoming evident. The intrathoracic nervous system acts as a distributive network, processing in a complex fashion information that arises not only from cardiac, vascular and pulmonary tissues but also from extrathoracic tissues, to maintain adequate cardiac function. In challenging the current understanding of cardiac regulation, this view provides novel opportunities to develop pharmacological and surgical strategies to manipulate cardiac function in disease states.

Ionic mechanism of inotropic effect of veratrine on frog heart

SummaryVeratrine alkaloids prolong the action potential and exert a positive inotropic effect in frog atrial muscle. In voltage-clamp experiments, veratrine sulphate slowed inactivation of the sodium system, thereby greatly increasing total sodium inward current and also tension; its effect on electrical and mechanical activity was blocked by tetrodotoxin. These findings indicate involvement of sodium ions in the inotropic action of veratrine alkaloids.

Diets enriched in menhaden fish oil, seal oil, or shark liver oil have distinct effects on the lipid and fatty-acid composition of guinea pig heart.

The purpose of this investigation was to determine whether diets supplemented with oils from three different marine sources, all of which contain high proportions of long-chain n-3 polyunsaturated fatty acids (PUFA), result in qualitatively distinct lipid and fatty acid profiles in guinea pig heart. Albino guinea pigs (14 days old) were fed standard, nonpurified guinea pig diets (NP) or NP supplemented with menhaden fish oil (MO), harp seal oil (SLO) or porbeagle shark liver oil (PLO) (10%, w/w) for 4-5 weeks. An n-6 PUFA control group was fed NP supplemented with corn oil (CO). All animals appeared healthy, with weight gains marginally lower in animals fed the marine oils. Comparison of relative organ weights indicated that only the livers responded to the diets, and that they were heavier only in the marine-oil fed guinea pigs. Heart total cholesterol levels were unaffected by supplementing NP with any of the oils, whereas all increased the triacylglycerol (TAG) content. The fatty-acid profiles of totalphospholipid (TPL), TAG and free fatty acid (FFA) fractions of heart lipids showed that feeding n-3 PUFA significantly altered the proportions of specific fatty-acid classes. For example, all marine-oil-rich diets were associated with increases in total monounsaturated fatty acids in TPL (p < 0.05), and with decreases in total saturates in TAG (p < 0.05). Predictably, the n-3 PUFA enriched regimens significantly increased the cardiac content of n-3 PUFA and decreased that of n-6 PUFA, although the extent varied among the diets. As a result, n-6/n-3 ratios were significantly lower in all myocardial lipid classes of marine-oil-fed guinea pigs. Analyses of the profiles of individual PUFA indicated that quantitatively, the fatty acids of the three marine oils were metabolized and/or incorporated into TPL, TAG and FFA in a diet-specific manner. In animals fed MO-enriched diets in which eicosapentaenoic acid (EPA) > docosahexacnoic acid (DHA), ratios of DHA /EPA in the hearts were 1.2, 2.2 and 1.5 in TPL, TAG and FFA, respectively. In SLO-fed guinea pigs in which dietary EPA ≈ DHA, ratios of DHA/EPA were 0.9, 3.4 and 2.1 in TPL, TAG and FFA, respectively. Feeding NP + PLO (DHA/EPA = 4.8), resulted in values for DHA/EPA in cardiac tissue of 2.1, 10.6 and 2.9 in TPL, TAG and FFA, respectively. In the TAG and FFA, proportions of n-3 docosapentaenoic acid (n-3 DPA) were equal to or higher than EPA in the SLO- and PLO-fed animals. The latter group exhibited the greatest difference between the DHA/n-3 DPA ratio in the diet and in cardiac TAG and FFA fractions (7, 3.4 and 3.1, respectively). Quantitative analysis indicated that ≥ 85% of the n-3 PUFA were in TPL, 7-11% were in TAG, and 2-6% were FFA. Specific patterns of distribution of EPA, DPA and DHA depended on the dietary oil. Both the qualitative and quantitative results of this study demonstrated that in guinea pigs, n-3 PUFA in different marine oils are metabolized and/or incorporated into cardiac lipids in distinct manners. In support of the concept that the diet-induced alterations reflect changes specifically in cardiomyocytes, we observed that direct supplementation of cultured guinea pig myocytes for 2-3 weeks with EPA or DHA produced changes in the PUFA profiles of their TPL that were qualitatively similar to those observed in tissue from the dietary study. The factors that regulate specific deposition of n-3 PUFA from either dietary oils or individual PUFA are not yet known, however the differences that we observed could in some manner be related to cardiac function and thus their relative potentials as health-promoting dietary fats.

Excitation—Contraction coupling in frog heart

SummaryThe mechanism by which veratrine alkaloids prolong action potential and exert a positive inotropic effect on frog atrial muscle was studied in voltageclamp experiments, using veratrine sulfate. Veratrine slowed the inactivation of Na conductance, thereby greatly increasing Na inward current; this current was suppressed by tetrodotoxin (TTX). The inward current increased in Na-free (lithium) but not in Na-free (sucrose). Ringers solution (Ri) after veratrine application. The positive inotropic effect was due to increased transmembrane movement of Na ions during electrical activity: when the influx of Na ions was abolished by TTX or the rate of electrical stimulation was suddenly decreased (<0.1 sec−1), the contractile force returned to control Ringer level after a short delay. The inotropic effect did not develop in Na-free (sucrose or LiCl) Ri. The data indicate that increased intracellular Na concentration [Na]i is responsible for regulating contraction during veratrines inotropic action.

Slow inward current and contraction in frog atrial muscle at various extracellular concentrations of Na and Ca ions.

Abstract NaCa antagonism in frog cardiac muscle was investigated under voltage-clamp conditions to obtain further information about the underlying mechanism(s). Specifically the relationship between the slow inward current ( I slow ) and that part of contractility activated by this current (phasic tension) was examined in various [Na] 0 and [Ca] 0 . When [Na] 0 was reduced by substituting with LiCl or sucrose I slow was changed only slightly (decreased in LiCl and increased in sucrose), whereas, phasic tension was increased several fold regardless of the type of substitute. When [Ca] 0 was reduced I slow was again less affected than phasic tension. This decrease in maximal peak tension resulting from reduced [Ca] 0 was partially but not fully compensated for by simultaneously decreasing [Ca] 0 and [Na] 0 , while keeping the ratio [Ca] 0 [Na] 0 2 constant. The current decreased almost immediately upon application of these low Na, low Ca solutions but the change in phasic tension was biphasic—an increase followed by a decrease. The results indicate that contractile force in frog myocardium does not simply depend on [Ca] 0 [Na] 0 2 . The NaCa antagonism is explained by an interaction between the slow inward current and the intracellular stores of Ca which are regulated by NaCa exchange. A qualitative model of regulation of cardiac contractility is presented.

Effects of chloride replacement in cardiac muscle mechanisms of positive inotropy

Abstract Replacement of chloride ions by various substitutes prolongs the cardiac action potential and has a positive inotropic effect (PIE) in frog myocardium. The underlying mechanisms of 99% chloride substitution by several anions (methanesulfonate, l -pyroglutamate, propionate, methylsulfate and nitrate) were analyzed in frog atrial muscle using a voltage-clamp technique. With all five chloride substitutes the amplitude of slow inward current was increased. This increase was TTX-insensitive (3 × 10−6 g/ml) and was present in Ca-free media but not in Na-free solutions. The PIE of chloride substitution was due to an increase in both components of contraction (phasic and tonic), and was absent in Na-free solutions. Furthermore, the positive inotropy was more pronounced at lower frequencies of stimulation and in the presence of bicarbonate than it was when Hepes was used. Various mechanisms that may be involved in PIE of chloride-substituted media are discussed.