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Cardiovascular Research 67 (2005) 225 – 233 www.elsevier.com/locate/cardiores

Peroxynitrite-induced a-actinin nitration and contractile alterations in isolated human myocardial cells

a

Division of Clinical Physiology, Institute of Cardiology, UD MHSC, P.O. Box 1, H-4004 Debrecen, Hungary b Department of Medical Chemistry, UD MHSC, P.O. Box 1, H-4004 Debrecen, Hungary c USZ Department of Pharmacology and Pharmacotherapy, P.O. Box 427, H-6701 Szeged, Hungary d Laboratory for Physiology, Institute for Cardiovascular Research, VU University Medical Center, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands Received 19 January 2005; received in revised form 11 March 2005; accepted 30 March 2005 Available online 26 April 2005 Time for primary review 27 days

Abstract Objective: Peroxynitrite-mediated myocardial protein nitration has been associated with a depressed cardiac pump function. In the present study, an attempt was made to elucidate the molecular background of peroxynitrite-evoked alterations in the human myocardium. Methods: Isometric force generation was measured in permeabilized human ventricular myocytes and biochemical methods were employed to identify the proteins affected by peroxynitrite-induced nitrotyrosine formation. Results: The maximal Ca2+-activated isometric force (pCa = 4.75) decreased to zero with increasing concentrations of peroxynitrite in a concentration-dependent manner (IC50: 55 T 4 AM; based on a total of 75 myocytes). However, there were no differences before and after the application of 50 AM peroxynitrite in the Ca2+-sensitivity of force production (pCa50: 5.89 T 0.02 and 5.86 T 0.04), in the steepness of the Ca2+-force relationship (nHill: 2.22 T 0.11 and 2.42 T 0.25), and in the actin – myosin turnover kinetics (k tr at saturating [Ca2+]: 1.14 T 0.03 1/s and 1.05 T 0.07 1/s) ( P > 0.05). Nevertheless, 50 AM peroxynitrite greatly deteriorated the cross-striation pattern and induced a slight, but significant, increase in the passive force component (from 2.1 T 0.1 to 2.5 T 0.2 kN/m2; n = 57 cells), reflecting ultrastructural alterations. Western immunoblots revealed that 50 AM peroxynitrite selectively induced the nitration of a protein with an apparent molecular mass of about 100 kDa. Subsequent immunoprecipitation assays identified this nitrated protein as a-actinin, a major Z-line protein. Conclusions: These results suggest a-actinin as a novel target for peroxynitrite in the human myocardium; its nitration induces a contractile dysfunction, presumably by decreasing the longitudinal transmission of force between adjacent sarcomeres. D 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved. Keywords: Myocytes; Contractile function; Peroxynitrite; a-Actinin; Human myocardium

This article is referred to in the Editorial by N. Paolocci (pages 176 – 178) in this issue. 1. Introduction Peroxynitrite-evoked protein alterations have been implicated in myocardial injuries that develop during reperfusion following ischaemia [1], or as a consequence * Corresponding author. Tel./fax: +36 52 414928. E-mail address: [email protected] (Z. Papp).

of exposure to inflammatory cytokines [2,3] or cardiotoxic drugs (e.g. doxorubicin) [4,5]. Moreover, increased levels of nitric oxide and reactive nitrogen species, e.g. peroxynitrite, may contribute to the development of congestive heart failure [6– 8]. Peroxynitrite modulated myocardial proteins via the formation of nitrotyrosine [9] and the amount of nitrated proteins correlated with the reduction in cardiac pump function in different animal preparations [3,4]. Nevertheless, the mechanism by which nitrated myocardial proteins decrease the myocardial contractile function in the human heart in particular remains obscure.

0008-6363/$ - see front matter D 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.cardiores.2005.03.025

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Attila Borbe´lya, Attila To´tha, Istva´n E´desa, La´szlo´ Vira´gb, Julius Gy Pappc, Andra´s Varro´c, Walter J. Paulusd, Jolanda van der Veldend, Ger J.M. Stienend, Zolta´n Pappa,*

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2. Methods 2.1. Myocardial tissue samples Healthy tissue was obtained from general organ donors whose hearts were also used for pulmonary and aortic valve

transplant surgery. These experiments complied with the Helsinki Declaration of the World Medical Association and were approved by the Albert Szent-Gyo¨ rgyi Medical University Ethical Review Board (No: 51 – 57/1997.OEj). Left ventricular wall samples were obtained from the base. All biopsies were stored in cardioplegic solution, composed of (in millimolar, mM) NaCl 110, KCl 16, MgCl2 1.6, CaCl2 1.2, and NaHCO3 5, and kept at 4 -C for ¨ 6 –8 h before being frozen in liquid nitrogen. Six donor hearts were used (3 men, 3 women; age = 39 T 5.1 years). The donors did not show any sign of cardiac abnormalities and did not receive any medication except for plasma volume expanders, dobutamine and furosemide. The cause of death included cerebral contusion due to accidents and cerebral haemorrhages or subarachnoidal haemorrhages due to stroke. The choice of tissue samples from individuals without known cardiovascular diseases minimized uncertainties related to tissue inhomogeneity. 2.2. Force measurements in single myocyte-sized preparations Frozen tissue blocks were first defrosted and mechanically disrupted in cell isolation solution (in mM: Mg2+ 1, KCl 145, EGTA 2, ATP 4, and imidazole 10; pH 7.0). The suspension was incubated in this solution supplemented with 0.3% Triton X-100 (Sigma, St. Louis, MO, USA) (5 min), washed and kept in cell isolation solution on ice for a maximum of 12 h. Subsequently, a demembranated single cardiomyocyte was mounted between two thin needles with silicone adhesive (Dow Corning, Midland, USA) while viewed under an inverted microscope (Axiovert 135, Zeiss, Germany) [18]. One needle was attached to a force transducer element (SensoNor, Horten, Norway) and the other to an electromagnetic motor (Aurora Scientific Inc., Aurora, Canada). The measurements were performed at 15 -C and the average sarcomere length was adjusted to 2.2 Am as described previously [19]. The compositions of the relaxing and activating solutions used during force measurements were calculated as described previously [16,20]. The pCa, i.e. log[Ca2+], values of the relaxing and activating solutions (pH 7.2) were 9 and 4.75, respectively. Solutions with intermediate free [Ca2+] levels were obtained by mixing activating and relaxing solutions. All the solutions for force measurements contained (in mM): Mg2+ 1, MgATP 5, phosphocreatine 15, and N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) 100. The ionic equivalent was adjusted to 150 with KCl at an ionic strength of 186. Isometric force was measured after the preparation had been transferred from the relaxing solution to a Ca2+-containing solution. When a steady force level was reached, the length of the myocyte was reduced by 20% within 2 ms and then quickly restreched (slack test). As a result, the force first dropped from the peak isometric level to zero (difference = total peak isometric force) and then started to redevelop. The force

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A decreased efficiency of the heart to utilize ATP for work has been described following the treatment of working rat hearts with peroxynitrite or cytokine [10,11], pointing to the contractile process as a potential mediator of the peroxynitrite-induced mechanical dysfunction. The peroxynitriteinduced reduction of myofibrillar Ca2+-responsiveness was found to be linked to the activation of the cGMP-dependent protein kinase pathway [12]. Alternatively, nitration of the 40 kDa myofibrillar isoform of creatine kinase was suggested as a mechanism responsible for the disturbed conversion of ATP to mechanical work in the hearts of doxorubicin-treated mice and the peroxynitrite-treated cardiac trabeculae of rats [4,13]. It is important that peroxynitrite-induced nitrotyrosine formation is not restricted to a single myofibrillar protein, either in animal or in human myocardial preparations [5,14,15]. Hence, the mechanical dysfunction will depend on the extent of tyrosine nitration in a set of affected myocardial proteins and on their functional and/or structural consequences. In this study we set out to elucidate the relationship between peroxynitrite-induced protein nitration and Ca2+activated force production in the human heart. Peroxynitriteelicited functional and structural alterations were monitored in permeabilized myocytes. The advantage of these preparations is that they present negligible diffusion obstacles [16] and hence allow almost instantaneous equilibration of peroxynitrite between the bathing medium and the myoplasmic space. Problems associated with the short lifetime of peroxynitrite were thereby minimized. The parameters of the Ca2+-force relationship considered were the force at saturating [Ca2+] ( P o), the Ca2+-sensitivity of isometric force production (pCa50), the steepness of the Ca2+-force relationship (nHill), the passive force component at a sarcomere length of 2.2 Am, and the cross-bridge specific rate constant of force redevelopment (k tr) [17]. These parameters were determined before and after exposure to peroxynitrite in order to reveal the effects of protein nitration on the mechanical function of the contractile system. Additionally, Western immunoblot analyses combined with immunoprecipitation assays were employed to pinpoint the contractile proteins directly responsible for the peroxynitrite-evoked contractile alterations. Our data indicated that the tyrosine nitration of a-actinin and the deterioration of the myofibrillar cross-striation pattern are tightly coupled to the reduction of isometric force production upon peroxynitrite exposure. This suggests that ultrastructural alterations may play a significant role in the peroxynitrite-mediated myocardial dysfunction in the human heart.

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2.3. Peroxynitrite administration The mechanical parameters of the permeabilized myocytes were measured before and after peroxynitrite exposure. Peroxynitrite (Cat. No. 516620) with low (¨ 0.1%) hydrogen peroxide content [21] was obtained from Calbiochem (San Diego, CA, USA). Before each experiment concentrated stock solutions of peroxynitrite (ranging from 10 AM to 10 mM) were prepared based on peroxynitrite concentration determination by absorbance measurements at 302 nm [21]. The pH in stock solutions was adjusted to 11 (by KOH) to oppose peroxynitrite decomposition and the ionic equivalent was set at 150 mM KCl to minimize alterations in the composition of relaxing solution during peroxynitrite exposures. A single volume of 20 Al from these stock solutions was rapidly introduced into a droplet (180 Al) of relaxing solution (pH 7.2, T = 20 -C), which surrounded each myocyte preparation in the mechanical setup. This approach resulted in nominal peroxynitrite concentrations ranging from 1 to 1000 AM, which decreased quickly because of spontaneous degradation (half-life: less than 3 s in this system). Peroxynitrite exposure was terminated following 60 s of incubation. A biochemical assay [22] indicated that approximately 47% of the applied peroxynitrite decomposed to nitrite and 53% to nitrate in relaxing solution. 2.4. Immunoprecipitation Human permeabilized ventricular myocytes were treated with different concentrations of active or 500 AM decomposed peroxynitrite and then homogenized in relaxing solution for the immunoprecipitation assays. The homoge-

nates were centrifuged at 10,000 rpm for 10 min and the supernatants were used for further experiments (immunoprecipitation assays or dot blot). For immunoprecipitation, the assay mixture (1 ml each) contained: 250 Ag protein of the tissue homogenate (treated with active or decomposed peroxynitrite), 2 Ag antibody (anti-a-actinin (Sigma, St. Louis, MO, USA)) or mouse IgG (Zymed Laboratories, San Francisco, CA, USA), 2 Al protease inhibitor cocktail (Sigma, St. Louis, MO, USA), and 20 Al protein A/G agarose resin (Santa Cruz, Santa Cruz, CA, USA) in relaxing solution. The mixtures were incubated at 4 -C overnight under continuous agitation. The resin-bound complexes were separated by centrifugation (1000 rpm, 1 min) and washed three times. The washed pellets were boiled in SDS sample buffer (Quality Biologicals, Gaithersburg, MD, USA) for 10 min and then subjected to Western immunoblotting. 2.5. Western immunoblot Nitrotyrosine formation and a-actinin levels in peroxynitrite-treated (1– 1000 AM) permeabilized myocyte preparations were assayed by Western immunoblotting following SDS-polyacrylamide gradient gel electrophoresis (6 –18% gradient gels with 20 Ag of protein homogenates in each lane). In parallel, the loading was visualized by silver staining [23], as described previously [24]. Following the electrophoresis step, myofibrillar proteins were transferred to nitrocellulose membranes, which were subsequently incubated with 5% non-fat dry milk and thereafter with primary antibodies (monoclonal anti-nitrotyrosine (Cayman Chemicals, Ann Arbor, MI, USA), dilution 1 : 5000; polyclonal anti-nitrotyrosine (Upstate, Charlottesville, VA, USA), dilution 1 : 7500; and anti-a-actinin (clone EA-53, Sigma, St. Louis, MO, USA), dilution 1 : 5000). For visualization, biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA, USA) and enhanced chemiluminescence (Amersham Biosciences, Uppsala, Sweden) were used. For the evaluation of the immunoprecipitated samples, the whole resin-bound complexes were separated on 10% SDS-polyacrylamide gels and subsequently subjected to Western immunoblotting. Some of the assays were combined with the removal of the bound antibody complexes (stripping). To this end, membranes were washed with stripping buffer (2 mM DTT, 2% SDS, 400 mM NaCl, and 20 mM Tris – HCl, pH 7.4) for 90 min at 60 -C. The membranes were then blocked and treated with primary and secondary antibodies as above. 2.6. Dot blot Volumes at 1 Al of all tissue samples prepared as indicated above and treated with 0– 500 AM peroxynitrite were dotted onto nitrocellulose membranes. The total protein amounts and nitrotyrosine levels in the homogenates were next tested with anti-a-actinin and anti-nitrotyrosine antibodies, as described for the Western immunoblots. Dot intensities were

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redevelopment after the restretch was fitted to a single exponential in order to estimate the rate constant of force redevelopment (k tr) at various [Ca2+] levels under both control (before peroxynitrite) and test conditions (after peroxynitrite). The passive force component was determined in relaxing solution following the Ca2+ contractures. The Ca2+-activated isometric force was calculated by subtracting the passive force from the total peak isometric force. The Ca2+-activated force at submaximal levels of activation was normalized to that at maximal activation so as to characterize the Ca2+-sensitivity of isometric force production. After the first maximal activation at pCa 4.75, resting sarcomere length was readjusted to 2.2 Am, if necessary. The second maximal activation at pCa 4.75 was used to calculate maximal isometric force ( P o). Cells were subsequently exposed to a series of solutions with intermediate pCa to construct the force – pCa relationship. At the end of this series, cardiomyocytes were re-exposed to pCa 4.75. If the generated force at this stage was less than 80% of the initial value, the measurement was discarded (average decrease in P o was to 71 T 0.04% in 10 discarded cells out of a total of 75).

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quantified from unsaturated recordings by densitometry, using custom-prepared software. Nitrotyrosine levels were expressed relative to the dot intensity resulting from 500 AM peroxynitrite exposure. 2.7. Data analysis The relation between force and pCa was fitted to a Hill equation:  nHill  nHill  2þ nHill  PCa ¼ Po Ca2þ Ca50 þ Ca

=

3. Results Panel A of Fig. 1 shows that peroxynitrite decreased the maximal isometric force production ( P o at pCa 4.75) in permeabilized human ventricular myocytes and that the reduction in force development was larger in myocytes

A 30 µM peroxynitrite

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C lyc l.a nti -N Mo T no cl. an t An i -N ti-α T -ac tin in

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Peroxynitrite concentration (µM) Fig. 1. (A) Original force recordings in different isolated permeabilized human cardiomyocytes during maximal Ca2+ activation (pCa = 4.75) before (solid lines) and after the application (dotted lines) of increasing concentrations of peroxynitrite. Control measurements were performed in the presence of degraded peroxynitrite (top left panel, 250 AM decomposed peroxynitrite). The protocol of length and [Ca2+] changes during force measurements is given schematically in the upper left panel. (B) Dot blot assays with poly- and monoclonal nitrotyrosine-specific (NT) primary antibodies illustrated increasing levels of nitrotyrosine formation in response to increasing concentrations of peroxynitrite in myocyte homogenates. (Identical protein loads were verified by monoclonal anti-a-actinin antibodies. Dot blot assays were performed in triplicate.) (C) The dose – effect relations of peroxynitrite on the maximal isometric Ca2+-activated force and on protein nitrotyrosine formation (means T S.E.M.) exhibited antiparallel concentration dependences in overlapping peroxynitrite concentrations. (Nitrotyrosine levels were expressed relative to the maximal staining intensities with poly- and monoclonal anti-nitrotyrosine antibodies, respectively, during the dot blot assays.).

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where P Ca is the steady-state force, P o is the steady isometric force at saturating Ca2+ concentration, the Hill coefficient

nHill is a measure of the steepness of the relationship, and Ca50 (or pCa50) is the mid-point of the relation. Values are given as means T S.E.M. for n myocytes obtained from at least 5 different hearts. Differences were tested by means of Student’s unpaired t-test at a level of significance of 0.05 ( P < 0.05).

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exposed to higher peroxynitrite concentrations. Incubation with decomposed peroxynitrite, on the other hand, had no effect on isometric force production (Fig. 1A, control). To confirm specificity, dot blot analyses with nitrotyrosinespecific monoclonal and polyclonal antibodies were employed in parallel. These assays revealed increasing

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levels of protein nitration in response to higher peroxynitrite concentrations (Fig. 1B). The results of statistical analyses of isometric force values (in 75 different myocytes) and nitrotyrosine levels (3 assays in myocyte suspensions) are illustrated in panel C of Fig. 1. The maximal isometric Ca2+activated force ( P o: 28 T 2 kN/m2) decreased to zero in a

Control 50 µM peroxynitrite 10 kN/m2

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Fig. 2. (A) Original force recordings measured before (solid lines) and after 50 AM peroxynitrite exposure (dotted lines) at maximal (pCa 4.75) and submaximal (pCa 5.8) levels of Ca2+ activation in the same cardiomyocyte. (B) The pCa – relative force relationship constructed from the force recordings indicated that 50 AM peroxynitrite decreased the maximal Ca2+-activated force by 45% (left panel) (data points are means T S.E.M., n = 32 – 65 myocytes). When force values measured at submaximal Ca2+ concentrations before and after 50 AM peroxynitrite treatment were normalized to their respective maxima (pCa – normalized force relationship; right panel), no significant differences could be observed between the Ca2+-sensitivity curves of isometric force production. (C) An amount of 50 AM peroxynitrite did not alter the [Ca2+]-dependence of the rate constant of tension redevelopment following unloaded shortening and restretch (k tr) (n = 25 myocytes). (D) The effects of 1-min long incubations with 50 AM peroxynitrite (PN), 50 AM peroxynitrite plus 1 mM urate, 1 mM hydrogen peroxide, 1 mM NaNO3 or 1 mM NaNO2 on relative P o in relaxing solution (from left to right, respectively; n = 5 – 7 myocytes). Asterisks indicate significant differences vs. control. (E) The cross-striation pattern of the myocytes was damaged by peroxynitrite. The representative example illustrates the cross-striation as viewed under the microscope before (left panel) and after (right panel) the application of 50 AM peroxynitrite in an isolated human myocyte preparation.

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pCa 4.75

10 s

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0 25

50 100 250 500

ynitrite (i.e. nitrite and nitrate) have a very limited role, if any in our system. Moreover, the prevention of force decrease by the peroxynitrite scavenger urate (1 mM) further supported that the observed changes in myocyte mechanics were the direct consequence of peroxynitrite. Next we attempted to identify the contractile proteins affected by nitrotyrosine formation and hence responsible for the decreased Ca2+-activated force production in peroxynitrite-treated human myocytes. SDS-polyacrylamide gel electrophoreses followed by Western immunoblot assays were employed to identify the molecular masses of proteins with nitrotyrosine residues (Fig. 3). Lower concentrations of peroxynitrite (25 –100 AM) induced the nitration of a single protein at a molecular mass of about 100 kDa. At higher concentrations (250 –500 AM) of peroxynitrite, additional proteins also underwent nitration as indicated by the intense immunoreactivity in a wide range of protein molecular masses above and below 100 kDa. Although identical amounts of proteins were applied in the assays (as verified by Western immunoblotting with anti-a-actinin following stripping (Fig. 3, right panel)), these latter proteins were not stained in the presence of 50 AM peroxynitrite. The molecular mass of the nitrated 100 kDa protein was similar to that of a-actinin. To verify that the nitrated protein was indeed a-actinin, an immunoprecipitation study was performed (Fig. 4). Myocardial protein homogenates were first incubated with active or decomposed peroxynitrite (500 0

25 50 100 250 500

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210 134 α-actinin

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40.6 32.2

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kDa

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Anti-α-actinin (after stripping)

Fig. 3. Sequential Western immunoblot analyses of peroxynitrite-treated human myocardial proteins. Peroxynitrite concentrations are depicted on the top and molecular weights are on the left. Membranes were first developed with anti-nitrotyrosine antibody (left panel) and subsequently with anti-a-actinin antibody (right panel) following stripping. A clear increase in the nitrotyrosine staining between peroxynitrite concentrations of 25 and 100 AM was apparent only at the 100 kDa protein level. The bands below 82 and 32.2 kDa did not exhibit any peroxynitrite concentration-dependence and are therefore not considered specific for peroxynitrite treatment. High peroxynitrite concentrations (250 – 500 AM) induced nitrotyrosine formation in several myofibrillar proteins in a wide range of molecular weights. Equal intensities of a-actinin staining were present at 100 kDa (3 independent assays provided identical results).

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range of peroxynitrite concentrations (IC50: 55 T 4 AM) in which the protein nitration level exhibited a dramatic increase. To elucidate the mechanistic background of peroxynitrite-induced contractile alterations, the [Ca2+]-dependences of force and of the rate of force redevelopment following unloaded shortening and restretch (k tr) were determined before and after 50 AM peroxynitrite exposure (Fig. 2). Peroxynitrite decreased isometric force at all Ca2+ concentrations studied (Fig. 2A and B, left panel). However, following force normalization to the respective maximum (Fig. 2B, right panel), the Ca2+-sensitivity curve before peroxynitrite treatment did not differ from that obtained after peroxynitrite application (pCa50: 5.89 T 0.02 and 5.86 T 0.04; nHill: 2.22 T 0.11 and 2.42 T 0.25; before and after 50 AM peroxynitrite, respectively ( P > 0.05)). Additionally, the cross-bridge specific kinetic parameter k tr did not change either at pCa 4.75 (k tr,max: 1.14 T 0.03 1/s and 1.05 T 0.07 1/s before and after 50 AM peroxynitrite) or at submaximal Ca2+ concentrations (Fig. 2C; P > 0.05). Nevertheless, the cross-striation pattern of the myocyte preparations deteriorated after 50 AM peroxynitrite treatment (Fig. 2E) and the passive force component increased from 2.1 T 0.1 to 2.5 T 0.2 kN/m2 (n = 57 cells; P < 0.05), suggesting ultrastructural damage. Control force measurements (Fig. 2D) suggested that non-specific effects due to hydrogen peroxide contamination or the by-products of perox-

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A Peroxynitrite Anti-α-actinin Mouse IgG

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Fig. 4. Identification of the 100 kDa nitrated protein as a-actinin by immunoprecipitation. (A) The nitrocellulose membranes were developed with an a-actininselective antibody following immunoprecipitation with anti-a-actinin. Incubations with the same amount of IgG as for the immunoprecipitation from mouse served as controls. The a-actinin-specific staining at 100 kDa illustrated the effective separation of a-actinin from other myocardial proteins in both peroxynitrite-treated (500 AM peroxynitrite) and peroxynitrite-untreated myocardial preparations. (B) After stripping, the same membranes as in A were developed with an antibody selective to nitrotyrosine and the staining at 100 kDa in the peroxynitrite-treated sample identified a-actinin as a target protein for nitration. (Protocols for incubations are given schematically above the registrations. IgG HC: heavy chain of IgG; IgG LC: light chain of IgG. Immunoprecipitation assays were performed in triplicate.).

AM). The samples were then divided into parts for a-actinin immunoprecipitation (with added a-actinin-specific antibody) and for the control (with the same amount of IgG from the same species). The efficiency of the immunoprecipitation was tested with a-actinin-selective antibody (Fig. 4A). In contrast with the control, the appearance of the specific immunostained bands at the level of a-actinin indicated that a-actinin was well separated from the other myocardial proteins both in the peroxynitrite-treated and in the peroxynitrite-untreated homogenates. Next the immunocomplexes were removed from the nitrocellulose membranes and the same membranes were stained with an antibody specific for nitrotyrosine. This procedure clearly identified the nitrated 100 kDa protein as a-actinin following peroxynitrite exposure (Fig. 4B).

4. Discussion The results of this investigation revealed a close inverse relationship between the extents of a-actinin nitration and Ca2+-activated force production in human myocyte-sized preparations. The nitration of a-actinin may therefore contribute to the cardiac dysfunction observed under conditions evoking increased peroxynitrite production [1 – 4,14,25 –27] in the human heart. Peroxynitrite induced structural, rather than regulatory, alterations in the contractile apparatus because the Ca2+sensitivity curve of force production (described by pCa50 and nHill) and the cross-bridge cycling rates (k tr) were not affected up to the IC50 value. Moreover, the reduction in maximal isometric force was tightly coupled to the deterioration in the cross-striation pattern and to a modest

increase in the passive force component. This implies that the peroxynitrite-induced contractile alterations can be explained by a reduction in the number of force-generating cross-bridges due to the diminished longitudinal transmission of force along the sarcomeres [28,29]. The human a-actinin molecule is a relatively tyrosine-rich (2.9% tyrosine) structural protein that is essential for maintenance of the Z-line and for the integrity of the sarcomeres [30,31]. It is fully conceivable, therefore, that the alterations caused in the conformation of a-actinin by its nitration are involved in the structural and consequently the functional alterations upon peroxynitrite exposure in these human myocardial preparations. It should be noted that the concentrations of the peroxynitrite mixtures applied in this study were in all probability higher than those expected to occur under pathophysiological conditions. Accordingly, exposure to lower concentrations of peroxynitrite for a prolonged period of time would have mimicked in vivo conditions better, but this is hampered by the short lifetime of peroxynitrite at physiological pH. Interpretation of the results of in vitro peroxynitrite treatments might be complicated by inadvertent hydrogen peroxide contamination and by unspecific effects of peroxynitrite by-products. Results of our control force measurements, however, argued against these possibilities and strongly suggested peroxynitrite specific effects on myocyte mechanics in our system. Peroxynitrite-induced cardiac protein nitration, myofibrillar thinning and irregular striation patterns have already been documented in doxorubicin-treated mice [5] and in the cardiac trabeculae of the rat following peroxynitrite exposure [13]. Interestingly, during immunogold electron microscopy, longitudinal sections from the ventricular wall tissue

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could explain the apparently distinct sequences in the peroxynitrite-sensitivities of the myofibrillar proteins in human and animal hearts. We have outlined here the mechanism by which peroxynitrite impairs Ca2+-dependent myofibrillar force generation in the human heart. However, the peroxynitriteevoked cardiac dysfunction may also depend on those additional peroxynitrite-sensitive processes that converge to the contractile function of the myocardium. Besides contractile protein nitration, these may include myofilament phosphorylation, Ca2+ transport systems and the energetic balance of the myocytes [7,12,34 –40]. Further studies are therefore required to elucidate the relative contributions of the affected regulators to the overall pump function during peroxynitrite-induced human cardiac pathologies.

Acknowledgements This study was supported in part by the research grants of the Hungarian Ministry of Health (ETT 239/2003) and by the Hungarian Scientific Research Fund (OTKA T35182).

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of the doxorubicin-treated animals demonstrated high gold particle densities indicative of nitrotyrosine staining around the Z-lines [5]. Moreover, similarly to our results, peroxynitrite incubation decreased the maximal Ca2+-activated force without giving rise to alterations in the Ca2+sensitivity of force production in the peroxynitrite-treated permeabilized cardiac trabeculae of the rat [13]. Although nitration of a number of myofibrillar proteins was observed in these animal models, the high levels of nitrotyrosine in the 40 kDa myofibrillar creatine kinase suggested that this was responsible for the peroxynitrite-elicited myofibrillar changes in Ca2+-activated contractile function [5,6,13]. The mechanical alterations observed in the human myocyte preparations in our study extend previous experimental findings on the peroxynitrite-modulated myofibrillar function in animal hearts. However, our mechanical and biochemical data led us to propose an alternative explanation for the mechanical dysfunction. In the range of peroxynitrite concentrations at which the isometric force was diminished, only the nitration of aactinin was observed. Similarly to others [13 – 15], we could additionally detect protein nitrotyrosine formation in several abundant proteins, though only after exposure to very high concentrations of peroxynitrite (250 – 500 AM). The molecular weights of these other proteins indicated possible nitration of the myosin heavy chain and of the myofibrillar isoform of creatine kinase. Nitration of these proteins and possibly others may therefore also contribute to the disappearance of force in response to high nominal concentrations of peroxynitrite. At lower concentrations (i.e. around the IC50 of peroxynitrite on the Ca2+-activated force), however, any significant inactivation of creatine kinase was ruled out by the mechanical observations on our human myocyte preparations. Inhibition of the myofibrillar creatine kinase would disturb the regeneration of MgADP to MgATP and hence slow down cross-bridge cycling [16,32,33]. However, following exposure to 50 AM peroxynitrite, no alteration in k tr or its Ca2+-sensitivity was observed. Hence, it is concluded that the contractile dysfunction seen at this peroxynitrite concentration is a consequence of structural alterations leading to a deteriorated cross-striation pattern, most probably through the nitration of a-actinin. The extent of protein nitration upon exposure to peroxynitrite correlated poorly with the levels of expression of certain myocardial proteins or with their tyrosine content in the rat cardiac trabeculae. This leads to tentative explanations based on the tertiary structure of proteins and their microenvironment and accessibility, which could modulate the susceptibility of the tyrosine residues to nonenzymatic nitration [13]. This line of reasoning prompts us to suggest that, within the complex geometry of the myofibrillar system, the nitration of a-actinin might be favored over that of other relatively tyrosine-rich molecules in the human heart. Thus, a-actinin may be a principal target in cardiac pathologies involving increased peroxynitrite production. Our data further suggest that species differences

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