Effect of Yttrium on Calcium-Dependent Processes in ... - Springer Link

ISSN 0022-0930, Journal of Evolutionary Biochemistry and Physiology, 2014, Vol. 50, No. 3, pp. 221—226. © Pleiades Publishing, Ltd., 2014. Original Russian Text © I.V. Shemarova, K.V. Sobol’, S.M. Korotkov, V.P. Nesterov, 2014, published in Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, 2014, Vol. 50, No. 3, pp. 196—200.

COMPARATIVE AND ONTOGENIC BIOCHEMISTRY

Effect of Yttrium on Calcium-Dependent Processes in Vertebrate Myocardium I. V. Shemarova, K. V. Sobol’, S. M. Korotkov, and V. P. Nesterov Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russia E-mail: [email protected] Received October 3, 2013

Abstract—Inoptopic effect of yttrium acetate (Y3+) on myocardium of the marsh frog Rana ridibunda and its effect on ion transport across the inner mitochondrial membrane (IMM) of rat heart was studied. Y3+ was found to decrease the rate of heart contractions and to stimulate ion transport in the rat heart mitochondria in media with 10 mM glutamate and 2 mM malate. Presence of Y3+ induced inhibition of energy-dependent Ca2+ transport into mitochondria, which was expressed as a marked decrease of their swelling in the media containing 125 mM NH4NO3 and Ca2+ or 25 mM potassium acetate, 100 mM sucrose and Ca2+. It is suggested that the Y3+-induced decrease in rat muscle contractions is determined not only by direct suppressing effect of Y3+ on potential-modulated Са2+-channels of pacemaker and contractile cardiomyocytes (CM), but also by its indirect effect on Са2+-carrier in IMM. The data confirming that Y3+ activates energy-dependent K+ transport catalyzed by mitochondrial uniporter and blocks Ca2+-channels in the mitochondrial membrane are important for more complete understanding of mechanisms of the Y3+ action on vertebrates and human CM. DOI: 10.1134/S0022093014030041 Key words: myocardium, inotropic effect, contraction, rare earth metals, Y3+, Са2+, mitochondria, swelling, vertebrates.

INTRODUCTION Rare earth metals (of cerium and yttrium groups) belong to the kind of substances whose biological role is studied insufficiently [1]. It is considered that the content of metals of this group in environment is rather low and therefore they cannot produce a considerable effect on human organism [2]. However, due to widening of sphere of application of rare earth metals in industry and medicine [3], the necessity to study their possible toxic effect on the cells and tissues of vertebrates appears to be important. The rare earth metals are antagonists

to Сa2+, which allows considering them as the possible competitive inhibitors of calcium channels localized in the plasma membrane of cells of different types including cardiac myocytes. High chemical activity of rate earth elements (REE), their capacity to form stable complex compounds and to react with inorganic cytoplasm components also are the basis for assumption of the possible toxic effect of REE on the cells. Among REE, the metals with lower ion radius are known to have the highest reactive capacity. In this connection, it is important to study effect on myocardium of the Y3+ ions occupying the intermediate position in

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the row between Sc3+ and Lu3+. In the earlier work we studied action of La3+ ions on cardiac muscle and CM mitochondria and revealed dependence between the La3+-induced mitochondria swelling and a decrease of the myocardium contraction rate [4]. Taking into account the differences in physic-chemical La3+ and Y3+ characteristics and scarce data on effect of Y3+ on properties of isolated mitochondria [5] and myocardium contraction [6], one would possibly suggest that comparison of data on effects of Y3+ and La3+, typical representatives of heavy and light REE, on CM allows obtaining a new knowledge on mechanisms of the REE toxic effects on excitable cells of vertebrate animals and humans. In the medical cardiology the diagnostics of functioning of the cardiovascular system (CVS) is one of important goals. Among parameters, characterizing CVS state on the whole and contractile capacity of myocardium in particular, are such parameters as heart rate, arterial and venous blood pressure, cardiac output [7]. However, in patients, determination of changes in myocardium contraction to the full degree is impossible. In experimental biology, changes in the heart contraction are traditionally evaluated in situ, by using isolated heart or preparations of heart fragments of homoiothermal or poikilothermal animals [8–10]. Application of methods allowing recording and analysis of contraction parameters, such as amplitude of contraction, duration of relaxation periods, development of muscle effort, etc., gives a possibility of clearly seeing action of the studied compounds on myocardium. Effect of Y3+ on myocardium contraction was not studied earlier, therefore we thought it important to study its inotropic effects in experiments in situ and to estimate its possible toxic effect on vertebrate and human CVS. At the cellular level, the most vulnerable targets for action of different damaging factors are mitochondria. This is confirmed by experimental data on action of oxidants and X-rays on permeability of mitochondrial membranes isolated from mouse liver and mitochondrial respiration [11, 12]. At the basis of damaging action of unfavorable, stress factors on mitochondria is their capacity to disturb mechanisms of ion transport via inner mitochondria membrane, what leads to changes

in mitochondrial volume and, hence, their photodiffusion. The changes in mitochondria light transparency is recorded spectrophotometrically [13], while the recording of changes in mitochondrial volume in Ca2+-containing medium allows evaluating changes in sensitivity of unspecific mitochondrial permeability in response to action of various factors including toxicants and X-rays [14]. Such method was successfully used for solving many tasks associated with evaluation of the functional state of mitochondria, in particular for studying their respiration and mechanisms of membrane permeability for examining action of various toxic substances on mitochondria [15, 16] or determination of the organism functional state under conditions of development of oxidative stress [11]. Therefore, one of the goals of the present investigation was to study effect of Y3+ on mitochondria by changes in their volume by using recording optical density of their suspension. The earlier performed studies have shown a possibility of correlation between the mitochondrial swelling and a decrease in inotropic effect [4], which allows using the data on rat mitochondria, with the results obtained on frog heart preparations. MATERIALS AND METHODS Recording and analysis of contraction parameters. To study the contraction properties, we used the muscle preparations representing a ring isolated from the middle part of heart ventricle of the marsh frog Rana ridibunda. The preparations were placed into a glass thermostatic chamber at 10°C and studied by our earlier described method [4]. The following parameters were estimated: the maximal effort developed by muscle preparations (Fmax, N; contraction amplitude), the period of effort development (tc, s), as well as the period of semi-relaxation (t1/2, s) of muscle preparation. Single spontaneous contractions of vertically oriented and preliminarily stretched preparations (by 150% from the initial length) were recorded under isometric regime. The standard incubation medium was the normal Ringer solution (g/l): 6.5 NaCl, 0.14 KCl, 0.1 СаCl2, 0.2 NaHСO3 (pH 7.4). This medium was used to prepare concentrated solution of yttrium ac-

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Fig. 1. Effect of Y3+ on values of isometric contraction of segments of frog heart ventricle. Spontaneous contractions in the normal physiological Ringer solution and solution with addition of 2 mM Y3+ (horizontal line under curve). Horizontal marker at above the right—time (s); vertical—strength (mN).

etate, Y(CH3COO)3 (Y3+), which was added in necessary volumes to the chamber for obtaining the designed final concentrations. The standard methods of statistical data processing were applied by using Student’s criterion. The differences were considered significant at p < 0.05. Isolation of mitochondria were applied. The mitochondria from rat heart were isolated on ice by our earlier described method [4]. The hearts of the male Wistar rats were minced, rinsed, and homogenized by using a polytron homogenizer in the medium containing (mM): 70 sucrose, 220 mannitol, 2 EGTA, 10 Tris-HCl (pH 7.3), and 0.1% bovine serum albumin (BSA). Then the tissue homogenate was centrifuged for 10 min at 400 g. Tissue sediment was kept on ice, while the supernatant containing mitochondria was centrifuged for 10 min at 8000 g. To increase yield of mitochondria, the homogenization procedure of tissue sediment with a potter homogenizer and subsequent centrifugation of tissues homogenate and supernatant was performed twice. The obtained mitochondria were rinsed twice in the medium containing 300 mM sucrose and 10 mM Tris-HCl (pH 7.3), and centrifuged for 10 min at 6500 g. Protein concentration in the mitochondrial preparations was determined by Bradford method [17] and was within 25–30 mg/ml. Spectrophotometrical evaluation of changes in

mitochondrial volume. The Y3+ effect on the IMM ionic permeability was performed on an SF-46 spectrophotometer at the wavelength of 540 nm by using the standard method [13] of mitochondrial swelling recorded at 20°C from a decrease in optical density of their suspension due to a fall of light dispersion. The mitochondria were suspended in the media (pH 7.3) containing (mM): 5 Тris-HCl and 125 NH4NO3 or 10 Тris-acetate, 25 CH3COOK and 100 sucrose. All media contained 2 μM rotenone and 1 μg/ml oligomycin. The protein concentration in the incubation medium was 1.0 mg/ml. Sucrose used in the work was purified from cation admixtures on the ion-exchange column with a KU-2-8 resin. 2,4-Dinitrophenol (DNP) and CaCl2 were of analytical grade, whereas mannitol, KH2PO4, CH3COOK и NH4NO3—of high purity grade. Glutamate, malate, Tris-OH, KCl, Y(CH3COO)3, АDP, EGTA, oligomycin and rotenone were purchased from Sigma (USA). The bovine serum albumin (BSA) used in the work was purified from fatty acids. RESULTS AND DISCUSSION Effect of Y3+ on amplitude of heart contractions. Inotropic effect of yttrium acetate was determined on isolated preparations of frog heart ventricle


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Fig. 2. Effect of Y3+ and Ca2+ on swelling of rat heart in salt media. Mitochondria (1 mg/ml of protein) were added to the medium containing 5 mM Tris-NO3 (pH 7.3) and 125 mM/l NH4NO3 (а) or 10 mM Tris-acetate, 25 mM CH3COOK and 10 mM sucrose (b). (1 ) Сontrol without additions, (2 ) experiment with addition of 33 μM Y3+; (3 ) experiment with addition of 150 μM Ca2+; (4 ) experiment with addition of 33 μM Y3+ + 150 μM Ca2+. Vertical arrows respectively indicate additions: rat heart mitochondria (RHM), 10 mM glutamate + 2 mM malate (10G + 2M). Horizontal axis: time (min); vertical axis: changes in optical density of mitochondrial suspension at the wavelength of 540 nm (ΔD).

under conditions of spontaneous excitation/contraction. The frog heart preparations produced the stable spontaneous contractions, lasting for a few

hours, in contrast to rat heart preparations. Y3+ ions at concentration of 2 mM in Ringer solution were found to decrease significantly the amplitude of spontaneous heart contractions (Fmax) by 65% (1.3 ± 0.14 mN in control; 0.46 ± 0.08 mN in experiment with yttrium acetate, p < 0.01) and to increase the period of semi-relaxation (0.25 ± 0.06 s in control, 0.47 ± 0.09 in experiment with yttrium acetate, p < 0.01) (Fig. 1). The period of development of effort increased insignificantly. It is known that the concentration of free calcium ions in cytosol, [Са2+]i, as well as the dynamics of its alterations inside the heart cell determine the maximally developing tension and the rate of contraction/relaxation [18]. In our experiments, Y3+ suppressed the contractility of myocardium, which is apparently a consequence of the decrease in the Са2+ concentration in the cytosol by application of yttrium acetate. An influx of extracellular Ca2+ into CM occurs predominantly through Ca2+-channels of L-type (Сa(v)1.2) [18]. Suppression of these channels with specific inhibitors (verapamil, nifedipine, etc.) leads to disturbances in the mechanism of triggering of contraction in the heart muscle [19], realized by the principle of Ca2+-induced Ca2+release from the sarcoplasmic reticulum (SR), as well as to a fall in the amplitude Fmax, and, as a result, to disturbance in heart activity. However, in the available literature we have failed data on mechanism of Y3+ effect both on calcium channels of plasma membrane in CM and on the CM contraction. It is suggested that in electro-excitable cells, the lanthanoids block influx Ca2+-currents activated by depolarization of membrane surface [20]. The comparison of effect of yttrium acetate and lanthanum chloride on parameters of CM contractions revealed similarity in inhibitory effect of these compounds, which can indicate the common mechanism of their blocking action on Сa(v)1.2. The Y3+ ions increased the time of semirelaxation of heart muscle, which might indicate a decrease in calcium reabsorption from the cytosol. The obtained results might indicate both possible Y3+ effect on the potential-regulated Ca2+-channels of contractive and pacemaker CM, which is manifested in the Fmax decrease, and its inhibitory action on the rate of Ca2+ reabsorption from the


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cytosol (an increase of t1/2). It cannot be ruled out that the negative inotropic effect of Y3+ might be determined not only by its competitive action on CM channel proteins and SR Ca2+-ATPase, but also by its direct toxic effect on CM mitochondria [4]. In this connection we thought it important to determine an effect of Y3+ on functional conditions of mitochondria and to establish its effect on cation transport via IMM. Effect of Y3+ on mitochondrial swelling. It is well accepted that IMM has low passive permeability, which might be evaluated by swelling of deenergized mitochondria in the media containing NH4NO3 [13]. The data presented on Fig. 2 show that in the presence of 33 μM Y3+ (curve 2), swelling of RHC de-energized mitochondria in isotonic medium with NH4NO3 remained the same as in experiments without Y3+ (curve 1). This indicates that Y3+ at the used concentration affects the passive proton permeability of IMM. Figure 2a demonstrates that the addition into incubation medium of Сa2+ (curve 3) regardless of the presence of Y3+ (curve 4) increased the swelling of de-energized mitochondria in the medium with NH4NO3, which might indicate stimulation by calcium of passive proton permeability of IMM. It is known that binding of Ca2+ ions to specific sites located in IMM from the matrix side under conditions of subsequent mitochondrial uncoupling can stimulate opening in this membrane of calciumdependent unspecific channel (CDUC) [21]. In our experiments the mitochondria energizing with glutamate and malate markedly stimulated their swelling in the medium with Ca2+ (curve 3), which is possibly due to opening of CDUC in this membrane. The presence of Y3+ in incubation medium (curve 4) substantially suppressed this process, which most likely is associated with inhibition by Y3+ of energy-determined Ca2+ transport via the mitochondrial Ca2+-uniporter. It is known that the mitochondrial swelling in the presence of oxidation substrates occurs predominantly as a result of accumulation of K+ cations in their matrix [22]. We evaluated an accumulation of K+ ions in the matrix of energized mitochondria in the experiments with application of hypoosmotic saline [13], containing 25 мМ CH3COOK (Fig. 2b). In this case swelling occurs due to energydependent absorption of K+ ions by mitochondria

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via K+-uniporter [13]. In parallel with this, mitochondria accumulate molecules of acetic acid, for which IMM is easily permeable. After dissociation of these molecules and subsequent pumping out of excessive protons from mitochondria, acetate anions are accumulated in the matrix. The performed investigations have shown that swelling of energized mitochondria in the medium with Y3+ occurs considerably faster than in control (Fig. 2b, curves 1 and 2) and can serve a confirmation of that Y3+ accelerates energy-dependent K+ transport into mitochondria, which is mediated by activation of the CM K+-uniporter. On the other hand, alkalization of the mitochondrial matrix and its swelling can also be induced by accumulation of other cations, including Ca2+ [23]. In this case, use of Y3+ as Ca2+ antagonist and CDUC blocker can result in suppression of Ca2+-dependent mechanisms of cation transport and a decrease in the mitochondrial swelling. To check this assumption, we performed an experiment with energized mitochondria in the above-mentioned hypoosmotic medium (Fig. 2b) containing Ca2+ (curve 3) or Ca2+ + Y3+ (curve 4). As seen from the data presented on this figure (curve 4), the mitochondrial swelling in the Y3+-containing medium was lower than in the medium with Ca2+ alone (curve 3). These results demonstrate an inhibitory action of Y3+ on the CDUC opening in IMM and can favor the presented suggestion. The data that Y3+ activates K+ transport through its uniporter in IMM and blocks Ca2+-channels of the mitochondrial membrane are important for the more complete understanding of mechanisms of the Y3+ effect in CM. Thus, the results obtained in the present work indicate an inhibitory effect of Y3+ ions on calcium-dependent processes in myocardium and can serve a confirmation of toxic action of yttrium acetate on organism of vertebrate animals and human. REDERENCES 1. Jalsaraeva, D.M., Side-effect of yttrium sulfate, Candidate Sci. Dissertation, Ulan-Ude, 2002. 2. Spasskii, S.S., Comparative toxicity of rare Earth metals, Gigiena Sanitariya, 1974, no. 4, pp. 33–36. 3. Turkmen, C., Kilicoglu, O., Dikici, F., Bezgal, F.,


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