AP08_1.vp Abbreviations NCX Na�/Ca2� exchanger SR sarcoplasmic reticulum SERCA SR Ca2� influx channel ECC excitation-contraction coupling NF non-failing heart F failing heart AM actomyosin complex 1 Introduction The number of known molecular of cardiac excitation- -contraction coupling (ECC) mechanisms is enormous and is still growing. It has become impossible to intuitively assess the relative contribution of an individual arrangement in physiological and pathophysiological events. However, such understanding is essential for identifying strategies for treat- ing various types of heart failure. Mathematical modeling and simulation offers a way of handling such an extensive set of data, while still allowing for a (semi)quantitative study. Of course, there are limitations to such an approach, mainly: the accuracy of the model, its complexity (always drastically lower than in reality), low availability of consistent experimental input data, etc. However, calculations and simulations have been already proven to be useful in biological research. As an example, let us mention the correct of Na-K exchanger stoichiometry 3:1, based on simulations prediction as early as 1985. [1] The aim of the present study is to assess the relative con- tribution of NCX and SERCA to the function of cardiac myocytes and thus to help interpret experimental data. Generally, both proteins reduce the cytoplasmic concen- tration of Ca2�, and influence both the systolic (contraction) and the diastolic (relaxation) function. While SERCA trans- ports Ca2� into the sarcoplasmic reticulum (SR) and so in- creases intracellular calcium stores, i.e. the systolic function, NCX removes Ca2� across the cellular membrane out of the cell (Fig. 1), so that the total cellular calcium is reduced and thus the systolic function is decreased. The role of NCX is far more complex, since it is regulated by voltage and by Ca2� and Na� concentration. Consequently, it can operate in so-called “reverse mode” during the early phase of action potential. Heart failure Congestive heart failure is an important medical issue with very high mortality and no targeted treatment avail- able. Though it is among the leading causes of death in our population, the underlying pathophysiological mechanisms at molecular level remain to be elucidated. Altered calcium (Ca2�) handling is seen as a key factor in the pathophysiolo- gy of heart failure. Typically, SERCA activity is consistently reported to be reduced in cases of advanced cardiac dysfunc- tion [2, 3]. The Na�/Ca2� exchanger (NCX) has also been reported to be altered in heart failure and the currently attracting strong interest [4]. The transporter may be over-ex- pressed [5] or normal or even reduced. The ratio between the two mechanisms seems to be an important determinant of overall function [2]. The model used for the present work is far from reproduc- ing all known mechanisms of ECC. However, it can still provide a valuable quantitative (or semi-quantitative) insight by providing calculated data that can help to decide which are the direct effects of NCX on cellular performance (under our © Czech Technical University Publishing House http://ctn.cvut.cz/ap/ 3 Acta Polytechnica Vol. 48 No. 1/2008 Computer Insight into the Molecular Level of Heart Failure. What is the Role of NCX? M. Fischer, M. Mlček, S. Konvičková, O. Kittnar Though congestive heart failure is a leading cause of death in our population, the pathophysiological mechanisms at molecular level remain to be elucidated. This paper discusses the contribution of NCX to the pathological pattern of intracellular calcium regulation and contraction on the basis of computer simulations of a virtual cell. The model comprises calcium handling mechanisms, troponin control and acto-myosin interactions. The contribution of NCX was studied by changing its activity and turning it off for some simulations. It was found that NCX helps to support diastolic function by reducing the Ca2� level during the diastole. At the same time there is a reduction in peak Cai and hence contraction. However, increased NCX activity does not seem to improve calcium handling and contraction crucially, as has been suggested by some authors. Keywords: heart failure, numerical models, calcium, NCX. Fig. 1: Some Ca2� transport mechanisms in heart cell limited conditions), and which findings cannot be attributed to NCX and require identification and/or the addition of other mechanisms. 2 Materials and Methods 2.1 Model An extended version of our earlier excitation-contraction model was used [6]. This model consists of gating, calcium, regulatory and contraction subunits (Fig. 2). A compart- mental description of the underlying molecular processes is adopted to simulate a) calcium handling, b) contraction con- trol by troponin, and c) the reaction kinetics of contractile elements. The contraction model and the calcium handling subunit were presented in [6] and [7]. Unlike the previous models, it also includes the Na�/Ca2� exchanger, which was omitted or extremely simplified in earlier versions. In principle, NCX current is now described after Winslow [8] as � � � � � � I t k t Na Ca e Na NaCa i e n r E t F RT e ( ) ( ) ( ) ( ) � � � � � � � � �3 2 3 � �Ca t ei n r E t F RT( ) ( ) ( ) ( ) � � � � � � 2 1 and ionic flux as Q t A C V z F I tNaCa cap SC c NaCa( ) ( )� � � � , where k(t) is the saturation parameter (see [8]), Nai (12 mol/m 3) is intracellular sodium concentration, Cae (1,8 mol/m3) is extracellular calcium concentration, n (3) is stoichiometry of Na-Ca exchange, r (0.15) is position of the peak of the energy barrier separat- ing two states – activation and deactivation of the exchanger, E(t) is action potential, F (96485.3415 s�A /mol) is Faraday constant, R (8.3144 J/K�mol) is ideal gas constant, T (290.15 K) is absolute temperature, Nae (140 mol /m 3) is extracellular sodium concentration, Cai(t) is intracellular calcium concentration, Acap (1.534 e 8 m2) is capacitive membrane area, CSC (1 F /m 2) is specific membrane capacity, Vc (25 e 12 m3) is cellular volume, and z (2) is valence of calcium ion. 2.2 Simulation and parameters The model was implemented in Matlab / Simulink. The ode15s (stiff/NDF) integration method was used. As an input, the experimentally obtained trace of cardiac action potential was used (Fig. 3) [9]. Simulation parameters: stimulation fre- quencies 1.2 and 3 Hz, simulation duration 300 s (to avoid transient state effects). The model was also verified for various action potential durations (APD) in accordance with reality where APD varies physiologically both with heart rate and in various regions of the myocardium. However, due to the simplicity, of the model, it currently does not essentially reflect any mecha- nisms influencing the shaping and action potential duration, such as modification of the activity of ionic channels. Heart failure model: Chronic congestive failure was simu- lated according to experimental findings [10] by reducing the activity of SERCA from 100% to 75 and 50 % of normal val- ues. SERCA ionic flux is defined by the equation Q t Ca t Ca k k f tiN i i iN iNact( ) ( ( ) ) ( ( ))� � � � �0 , where QiN(t) is ionic flux from the intracellular space to the net- work sarcoplasmic reticulum (NSR), 4 © Czech Technical University Publishing House http://ctn.cvut.cz/ap/ Acta Polytechnica Vol. 48 No. 1/2008 E t( ) Fig. 2: Model scheme – subunits interaction Fig. 3: Action potential – model input Cai0 (1 e 4 mol /m3) is initial intracellular calcium concentration, kiN (4 s 1) is the rate constant of the passive part of SERCA channel, kiNact (1 s 1) is the rate constant of the active part of the SERCA channel, and f�(t) is steady-state deactivation coefficient. According to some findings that report NCX activity in failing hearts [11], the NCX activity was varied between 50, 75, 100 and 130 % of normal activity. The NCX transporter was also virtually completely blocked (activity set to 0 %), so that its (missing) role in ECC could be identified more easily. 3 Results 3.1 NCX in a non-failing heart (NF) Though NCX is a minor calcium-removal mechanism (10~25 % of total Ca to be removed), its contribution to calcium handling is substantial and becomes obvious over a period of time (here, after 300 s at 1 Hz). The absence of NCX activity (Fig. 4) results in more Ca2� remaining in the cell. This calcium is transported into internal stores (the sarcoplasmic reticulum) by the SERCA transporter. Even the rather small amount of Ca2� that is retained during each beat gradually loads the internal calcium stores until a new input-output © Czech Technical University Publishing House http://ctn.cvut.cz/ap/ 5 Acta Polytechnica Vol. 48 No. 1/2008 Cai (F) 0 00E+00. 1 00E-04. 2 00E-04. 3.00E-04 299 00. 299 25. 299 50. 299 75. 300 00. time (s) C a i (m o l) norm no NCX NCX 150 Fig. 4: Intracellular Ca2� concentration in NF and F heart (NCX activity 0 % and 150 %), at 1 Hz AM 0 00E+00. 5 00E-09. 1 00E-08. 1 50E-08. 2.00E-08 299 00. 299 25. 299 50. 299 75. 300 00. time (s) A M (m o l) norm no NCX Fig. 5: Contractile force in NF heart and in heart with turned NCX off, at 1 Hz equilibrium is reached. Thus during activation more Ca2� will be released and so more force will be generated. At the same time, the relaxation of contraction is prolonged. Increased NCX activity exhibits opposite effects. Thus it can be concluded that increased NCX activity (the forward mode) alone decreases the force (negative inotropic effect) and improves relaxation (positive lusitropic effect), and vice versa. The kinetics of an acto-myosin strong interaction (AM(t)) represents the mechanical performance of virtual cardiac myocyte. This is obtained from the model simulation (con- traction subunit). The original description was given in [6]. The effect of heart rate The effect of NCX during each cardiac cycle is time-lim- ited, and is therefore directly influenced by heart frequency. Shortening the cardiac period means that less time is avail- able for Ca2� removal by NCX (Fig. 8). If NCX activity is decreased, at higher rates (2–3 Hz) Ca2� does not return to 6 © Czech Technical University Publishing House http://ctn.cvut.cz/ap/ Acta Polytechnica Vol. 48 No. 1/2008 AM, 3 Hz 0 00E+00. 5 00E-09. 1 00E-08. 1 50E-08. 2 00E-08. 2.50E-08 299 00. 299 25. 299 50. 299 75. 300 00. time (s) A M (m o l) norm no NCX Fig. 6: Contractile force in NF heart and in heart with turned NCX off, at 3 Hz Ca fluxes at 1 Hz, normal cells 0 00E+00. 4 00E-03. 8 00E-03. 1.20E-02 1.60E-02 QiE QiN Q NCX C a fl u x e s (m o l) Fig. 7: Total Ca2� fluxes (per 1 sec) at 1 Hz, NF the baseline, and therefore the myocardium may not relax properly between beats (Fig. 6). 3.2 NCX in SERCA dysfunction Impaired calcium handling in chronic heart failure (F) was simulated by decreasing the SERCA activity to 50 % of the norm. The activity of NCX was varied between 0, 100 and 150 %. Decreased SERCA activity alone resulted in lower calcium peak and force transient (by cca 40%) and increased calcium during the relaxation phase (compared to the non- -failing model). This is consistent with many experimental findings [11, 12]. By increasing the activity of NCX to 150 % of N, the relaxation (force and Ca2�) was restored. However, this is at the cost of a further decline of the maximal force (to 50 %) (Fig. 9). Importantly, this effect was pronounced at high frequencies (3Hz – Fig. 10). 4 Discussion The role of NCX in ECC is quite complex. At first, by removing calcium from the cell, increased NCX reduces © Czech Technical University Publishing House http://ctn.cvut.cz/ap/ 7 Acta Polytechnica Vol. 48 No. 1/2008 Q NCX (norm) 0.00E+00 2.10E-03 1 Hz 2 Hz 3 Hz Q N C X (m o l/ s ) Fig. 8: Total Ca2� fluxes (per 1 sec) at 1, 2 and 3 Hz, NF Cai (in NF and F) 0 00E+00. 2.10E-03 299 00. 299 25. 299 50. 299 75. 300 00. time (s) C a i (m o l) norm F, NCX 100 F, NCX 150 Fig. 9: Intracellular Ca2� concentration in NF and F heart (SERCA activity 50 %, NCX activity 100 % and 150 %), at 1 Hz contractility and thus could have a negative effect on cardiac performance. This is in line with some experimental findings [13]. Though the transporting capacity of NCX is much lower than that of other mechanisms (SERCA), NXC can influ- ence the gradual loading/unloading of cellular calcium stores. Cellular calcium determines the maximal contraction force. Secondly, lowering cellular calcium (both in stores and free Ca2�) has a direct positive effect on cardiac relaxation and so can improve heart performance – the heart is a pulsation pump so the relaxation is as necessary as the contraction and both need to alternate periodically. Moreover, the nutrition (blood supply) of the heart is better during the relaxation phase (it almost stops during peak contraction). Thus it can- not be simply concluded which of the two effects should take precedence. Nor can we calculate the amplitude of force (AM) oscillation as a measure of performance. Thirdly, NCX is capable of “reverse mode” operation, i.e. it can load calcium into the cell during the early phase of the cardiac cycle. The effect of NCX is also heart-rate dependent. At low frequencies (<~1Hz), the relatively long period offers enough reserve capacity for extrusion of Ca2�, which controls relaxation. The extrusion (and therefore the relaxation) takes longer, but ultimately fully completes (Figs. 4, 5). At higher rates full re- laxation is not achieved. 8 © Czech Technical University Publishing House http://ctn.cvut.cz/ap/ Acta Polytechnica Vol. 48 No. 1/2008 AM / FORCE (in NF and F), 3 Hz 0 00E+00. 2 00E-09. 4 00E-09. 6 00E-09. 8.00E-09 299 00. 299 25. 299 50. 299 75. 300 00. time (s) A M (m o l) norm F, NCX 100 F, NCX 150 Fig. 10: Contractile force in NF and F heart (SERCA activity 50 %, NCX activity 100 % and 150 %), at 3 Hz Ca fluxes (per cycle), 1 Hz 0 00E+00. 4 00E-03. 8 00E-03. 1 20E-02. 1.60E-02 QiE QEi QiN QNCX Q (m o l/ s ) norm F, NCX 100 F, NCX 150 Fig. 11: Total Ca2� fluxes (per 1 sec) in NF and F heart (SERCA activity 50 %, NCX activity 100 % and 150 %), at 1 Hz In heart failure, reduced SERCA is consistently reported. In line with this, lower total calcium, calcium peak and maxi- mal force are found. However, reports on the role of NCX are almost contradictory. O’Rourke [11] reports increased NCX that compensates defective SERCA in maintaining diastolic (relaxed) calcium low at the cost of further compromised sys- tolic (contraction) performance. Based on our results, this compensatory effect is relevant mainly when the heart rate is higher. However, Hobai [12] reports improved cardiac function in a failing heart after inhibition of increased NCX by externally administered inhibitory peptide, and suggests NCX inhibi- tion as a promising treatment for heart failure. This finding may also be relevant, however only when the heart rate is low (circa 1 Hz and less). Blocking NCX: a) increases cellular cal- cium loading (and contractility), b) increases diastolic Ca2�. At low heart rates, there is enough time to extrude this increased Ca2� (Fig. 9) and so maintain relaxation. At low rates overall stimulation predominates. At rates � 2 Hz diastolic Ca2� ac- cumulates, since it is not sufficiently extruded and results in incomplete relaxation (Fig. 10). The experiments by Hobai were performed at 0.5 Hz which may explain why they reported a generally beneficial role of NCX inhibition. How- ever, in clinical cases there could be double-edged effects, since the heart frequency can easily reach 2 Hz. Some other works have suggested increased cardiac re- serve performance after over-expression of NCX in a failing rabbit heart [13]. This effect cannot be explained by the current model, and thus requires further mechanisms to be identified and included. No matter how complex calcium handling is, it must be true that the input and output of Ca2� into and out of the cy- toplasm must generally match. Any change in one of the transporting mechanisms (SERCA, NCX, others) must be re- flected by changes in other parameters (cellular Ca2� stores, free Ca2�i, etc.) The major role of NCX is in removing Ca 2�, which (necessarily) enters the cell through an L-type channel (DHPR) from outside during each cycle. Thus increased NCX activity will always tend to unload the cell and compromise heart contractility. Increased NCX 200 % combined with re- duced SERCA 50 %, as sometimes reported in heart failure, would result in extreme depletion of internal calcium and thus the force that could be generated is hardly sufficient. This calculation indirectly suggests that another mechanism must be involved in Ca2� handling changes in heart failure. One option is the “reverse mode” capability of NCX in the early phase of a cycle. This regime depends on many parame- ters (Na� and Ca2� concentrations, membrane voltage, NCX regulations) and plays a minor role under physiological con- ditions. However, to make a quantitative assessment of this effect and its contribution to cell Ca2� handling as a whole, specifically in a simulated failing heart, the current model needs to be refined to better reflect the parameters control- ling the NCX reverse mode. Limitations In biological systems numerous molecular mechanisms contribute to ECC. Any of them can be altered and many may even not have been identified yet. Conversely, a computa- tional model can only reflect a very limited set of the mecha- nisms that are included in the real phenomenon in real life. The results drawn here should always be interpreted with caution. However, good conformance with experimental da- ta indicates that major mechanisms have been identified (e.g. the effect of NCX at various heart rates). However, the discrepancy between the simulated and experimental data suggests that further mechanisms need to be searched for (e.g. improved contractility after NCX stimulation in healthy hearts). In this way, simulations can stimulate and direct bio- medical research, which has traditionally relied on experi- mental approaches. 5 Acknowledgment This study was supported by the Ministry of Education, Youth and Sports of the Czech Republic project: Transdisci- plinary research in Biomedical Engineering II., No. MSM 6840770012 and the Grant Agency of the Czech Republic, project No. 106/04/1181, and Czech Academy of Sciences 1ET201210527. References [1] Di Francesco, D. – Noble, D.: A Model of Cardiac Elec- trical Activity Incorporating Ionic Pumps and Concen- tration Changes – Simulations of Ionic Currents and Concentration Changes. Philos.Trans. R. Soc. Lond B Biol. Sci., 1985, vol. 307, p. 353–398. [2] Hasenfuss, G. et al.: Relationship between Na�- Ca2�- ex- changer Protein Levels and Diastolic Function of Failing Human Myocardium. Circulation, 1999, vol. 99, p. 641–648. [3] Huke, S. et al.: Altered Force-Frequency Response in Non-Failing Hearts with Decreased SERCA Pump-Level. Cardiovasc. Res., 2003, vol. 59, p. 668–677. [4] Hasenfuss, G. – Schillinger, W.: Is Modulation of So- dium-Calcium Exchange a Therapeutic Option in Heart Failure? Circ.Res., 2004, vol. 95, p. 225–227. [5] Pieske, B. et al.: Functional Effects of Endothelin and Regulation of Endothelin Receptors in Isolated Human Nonfailing and Failing Myocardium. Circulation, 1999, vol. 99, p. 1802–1809. [6] Mlček, M. et al.: Mathematical Model of the Electro- mechanical Heart Contractile System—Regulatory Sub- system Physiological Considerations. Physiol Res., 2001, vol. 50, p. 425-432. [7] Novak, V. – Neumann, J.: Mathematical Model of the Electromechanical Heart Contractile System – Simula- tion Results. International Journal of Bioelectromagnetism, 2000, vol.2, no. 2, electronic version. [8] Winslow, R. L. et al.: Mechanisms of Altered Excita- tion-Contraction Coupling in Canine Tachycardia-In- duced Heart Failure, II: Model Studies. Circ.Res., 1999, vol. 84, p. 571–586. [9] Trautwein, W. – Kassebaum, D. G.:. Electrophysiological Study of Human Heart Muscle. Circ Res, 1962, vol. 10, p. 306–312. [10] Beuckelmann, D. J. – Nabauer, M. – Erdmann, E.: Intracellular Calcium Handling in Isolated Ventricular Myocytes from Patients with Terminal Heart Failure. Circulation, 1992, vol. 85, p. 1046–1055. © Czech Technical University Publishing House http://ctn.cvut.cz/ap/ 9 Acta Polytechnica Vol. 48 No. 1/2008 [11] O’Rourke, B. et al.: Mechanisms of Altered Excita- tion-Contraction Coupling in Canine Tachycardia-In- duced Heart Failure, I: Experimental Studies. Circ.Res., 1999, vol. 84, p. 562–570. [12] Hobai, I. A. – Maack, C. – O’Rourke, B.: Partial Inhi- bition of Sodium/Calcium Exchange Restores Cellular Calcium Handling in Canine Heart Failure. Circ.Res., 2004, vol. 95, p. 292–299. [13] Munch, G. et al.: Functional Alterations after Cardiac Sodium-Calcium Exchanger Overexpression in Heart Failure. Am. J. Physiol Heart Circ.Physiol, 2006, vol. 291, p. H488–H495. Martin Fischer, MSc. phone: +420 224 352 542 e-mail: mail@martinfischer.cz Department of Mechanics, Biomechanics and Mechatronics Czech Technical University Faculty of Mechanical Engineering Technická 4 166 07 Prague 6, Czech Republic Mikuláš Mlček, MD, Ph.D. phone: +420 224 968 407 e-mail: mikulas.mlcek@lf1.cuni.cz Institute of Physiology Charles University First Faculty of Medicine Albertov 5 128 00 Prague 2, Czech Republic Svatava Konvičková, MSc, Ph.D. phone: +420 224 352 511 e-mail: svatava.konvickova@fs.cvut.cz Department of Mechanics, Biomechanics and Mechatronics Czech Technical University Faculty of Mechanical Engineering Technická 4 166 07 Prague 6, Czech Republic Otomar Kittnar, MD, Ph.D. phone: +420224912903 e-mail: otomar.kittnar@ lf1.cuni.cz Institute of Physiology Charles University First Faculty of Medicine Albertov 5 128 00 Prague 2, Czech Republic 10 © Czech Technical University Publishing House http://ctn.cvut.cz/ap/ Acta Polytechnica Vol. 48 No. 1/2008 << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /None /Binding /Left /CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Error /CompatibilityLevel 1.4 /CompressObjects /Tags /CompressPages true /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /DetectCurves 0.0000 /ColorConversionStrategy /CMYK /DoThumbnails false /EmbedAllFonts true /EmbedOpenType false /ParseICCProfilesInComments true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams false /MaxSubsetPct 100 /Optimize true /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveDICMYKValues true /PreserveEPSInfo true /PreserveFlatness true /PreserveHalftoneInfo false /PreserveOPIComments true /PreserveOverprintSettings true /StartPage 1 /SubsetFonts true /TransferFunctionInfo /Apply /UCRandBGInfo /Preserve /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /CropColorImages true /ColorImageMinResolution 300 /ColorImageMinResolutionPolicy /OK /DownsampleColorImages true /ColorImageDownsampleType /Bicubic /ColorImageResolution 300 /ColorImageDepth -1 /ColorImageMinDownsampleDepth 1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages true /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /CropGrayImages true /GrayImageMinResolution 300 /GrayImageMinResolutionPolicy /OK /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /CropMonoImages true /MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /OK /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile () /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False /CreateJDFFile false /Description << /ARA /BGR /CHS /CHT /CZE /DAN /DEU /ESP /ETI /FRA /GRE /HEB /HRV (Za stvaranje Adobe PDF dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. 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