Inaugural Dissertation

Transcription

1 MYOSIN HEAVY CHAIN (MHC) ISOENZYMES: A NEW METHOD FOR ANALYSIS AND ASPECTS OF THEIR HETEROGENEOUS DISTRIBUTION IN NORMOXIC AND POSTISCHEMIC HEARTS Inaugural Dissertation Zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf vorgelegt von Solange Cristina Garcia Pomblum aus Mandaguaçu PR, Brasilien Düsseldorf 2002

2 Die vorliegende Dissertation wurde am Zentrum für Operative Medizin I an der Heinrich-Heine-Universität Düsseldorf in der Forschungsgruppe Experimentelle Chirurgie in der Zeit von Januar 2000 bis April 2002 angefertigt. Gedruckt mit Genehmigung der Mathematisch-Naturwissenschaflichen Fakultät der Heinrich-Heine-Universität, Düsseldorf, Deutschland Referent: Prof. Dr. Jochen D. Schipke Koreferent: Prof. Dr. Rudolf Bauer Tag der mündlichen Prüfung: 8. Juli 2002

3 Berichte aus der Pharmazie Solange Cristina Garcia Pomblum Myosin Heavy Chain (MHC) Isoenzymes: A New Method for Analysis and Aspects of their Heterogeneous Distribution in Normoxic and Postischemic Hearts. Gedruckt mit Unterstützung des Deutschen Akademischen Austauschdienstes D 61 (Diss. Universität Düsseldorf) Shaker Verlag Aachen 2002

4 Die Deutsche Bibliothek - CIP-Einheitsaufnahme Garcia Pomblum, Solange Cristina: Myosin Heavy Chain (MHC) Isoenzymes: A New Method for Analysis and Aspects of their Heterogeneous Distribution in Normoxic and Postischemic Hearts / Solange Cristina Garcia Pomblum. Aachen : Shaker, 2002 (Berichte aus der Pharmazie) Zugl.: Düsseldorf, Univ., Diss., 2002 ISBN Copyright Shaker Verlag 2002 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior permission of the publishers. Printed in Germany. ISBN ISSN Shaker Verlag GmbH P.O. BOX D Aachen Phone: 0049/2407 / Telefax: 0049/2407/ Internet: info@shaker.de

5 The important thing in science is not so much to obtain new facts as to discover new ways of thinking about them. Sir William Bragg

6 Table of contents I Table of Contents Page Table of contents...i Abstract... IV 1. Introduction Literature review Contractile proteins Myocardial stunning and oxidative effects Antioxidants Materials and methods Reagents and apparatus Optimization of minigel electrophoresis method Basis of electrophoresis method Tissue preparation Electrophoresis Staining/destaining and computer analysis Western blotting Reproducibility Heterogeneous distribution of myosin isoenzymes in rabbit hearts Normoxic hearts Age-dependency The transmural isoenzyme distribution ATP determination Ischemic hearts...42

7 II Table of contents 3.4 Antioxidants effects on myocardial function in postischemic hearts Global cardiac function Antioxidants effects on biochemistry markers and global myocardial... function Global cardiac function Myosin isoenzymes analysis ATP, troponin I, creatine kinase and lactate Statistic analysis Results Optimization of gel electrophoresis method Electrophoresis conditions Staining and destaining Western blotting Reproducibility Myosin isoenzyme distribution in rabbit myocardium Age-dependent distribution Transmural distribution of the myosin isoenzymes in rabbit... hearts Normoxic and ischemic hearts Effect of antioxidants on myocardial function in postischemic hearts Effect of antioxidants on biochemistry markers and myocardial... function in postischemic hearts Discussion Critique of methods New, nongradient nondenaturing PAGE method Age dependency and transmural distribution Normoxic and postischemic myocardium Oxidants and antioxidants effects on myocardial function Protective effects on myocardial function and protein alterations...103

8 Table of contents III 6. Summary and conclusion References Abbreviations List of figures List of tables...129

9 IV Abstract Abstract Myosin importantly contributes to structure and function of the cardiac muscle. Three isoenzymes of myosin heavy chain (MHC) V 1, V 2, and V 3 with different myosin ATPase activities have been identified in mammalian cardiac ventricles. The relative amount of myosin isoenzyme changes under physiological and pathological conditions. It is well established that reperfusion after brief, reversible myocardial ischemia is associated with prolonged depression of contractile function and a variety of ultrastructural, metabolic, electrophysiological, and other abnormalities. The identification of proteins modified during ischemia/reperfusion is fundamental to help understanding the pathological processes involved. Changes in the expression of cardiac MHCs resulting from different stimuli are traditionally evaluated by separating the myosin enzymes in nondenaturing gels. Isomyosins have also been determined using either nondenaturing tube gel PAGE or gradient or nongradient SDS-PAGE. These methods have particular shortcomings, e.g. long run times. This study reports a fast and efficient method to monitor myosin isoenzymes using a nongradient nondenaturing system in minigel format with Coomassie blue staining. After the method was established, the well known age-dependent effects and the unknown effects of myocardial ischemia on the distribution of cardiac myosin isoenzymes of rabbits were investigated. This new system permits analysis of the MHC isoenzymes in native conditions and better evaluation of the effects of physiological and pathological stimuli. The staining and destaining is easy and rapid. Multiple samples can be analysed under identical conditions, in contrast to tube gels that permit analysis of only one sample of a given gel. The agedependency shows in the adult hearts a predominance of V 3 over V 1, i.e. - MHC. In the juvenile heart a predominance of V 1 over V 3, i.e. -MHC was demonstrated. Ischemic hearts show an increase in V 1 (~70%), total loss of native V 2, and partial loss of V 3 (~30%) compared with normoxic hearts. Because of these deleterious changes, antioxidant effects of mannitol and glutathione on myocardial function and on protein damage were studied in isolated blood perfused rabbit hearts. Compared with placebo, both anti

10 Abstract V oxidants provide protective effects on ventricular function and help preventing protein damage. A correlation between myosin isoenzyme distribution, plasma proteins (creatine kinase and troponin I), and cardiac output was found: A reduction of protein damage in myocardium or in plasma corresponds to an improved myocardial dysfunction. The beneficial effects of both antioxidants suggest that oxidative stress greatly contributes to postischemic dysfunction.