Molecular response of porcine myocardium to ischemia and reperfusion

J Mol Cell Cardiol 24 (Supplement I) (1992) O-21-5 SODIUM ION-MEDIATED REGULATION OF Na,K-ATPase GENE EXPRESSION IN CARDIOCYTES Keiji Yamamoto, Uichi Ikeda, Yoshio Tsuruya, Asahiko Oguchi, Yoshitane Seine, Kiyoshi Kawakami*, Kei Nagano*, Yukichi Hara**, Kazuyuki Shimada. Departments of Cardiology, and *Biology, Jichi Medical School, Tochigi, **Department of Biochemistry, Tokyo Medical and Dental College, Tokyo, Japan. The purpose of this study is to determine whether Na’ regulates the levels of mRNA coding for Na,K-ATPase a and B subunits in primary cultured neonatal rat cardiocytes (NRC). We performed Nortbem blot analysis using Na,K-ATPase a and B isofonn specific cDNA probes. (i) Exposure of NRC to ImM ouabain, which increases intracellular Nd levels, resulted in 3-, 2-, and 5fold increases in Na,K-ATPase cc,, a, and Q, mRNA accumulation, respectively, with a peak elevation at 60 min. (ii) Exposure of NRC to low K’ (0.8mM) also caused a 3-fold increase in the a, mRNA accumulation with a peak elevation at 60 min. (iii) Transfection experiments by the calcium phosphate coprecipitation with cbimerlc plasmids containing S-flanking sequences of each a isoform gene and luciferase reporter gene revealed that 1mM ouabain caused an approximately 2-3-fold increase in the luciferase activity in each a isoform system. These results demonstrate that Na’ directly regulates Na+,K-ATPase gene expression in cardiocytes. The transfection study further supports the premise that Na response elements are located within the S-flanking sequences of each a isofonn gene.

O-21-6 MOLECULAR RESPONSE OF PORCINE MYOCARDIUM TO ISCHEYIA AND REPERFUSION Thomas Brand’, Han S Sharmat, Kirsten E Fleischmannl, Dirk J DunckeP, Edward McFalls*, Pieter D Verdouw*, Wolfgang Schaper 1; ‘Max-Planck-Institute, Exp. Cardiology, Bad Nauheim, FRG. *Exp. Cardiology Laboratory, Erasmus University, The Netherlands. The molecular basis of myocardial adaptation to &hernia and reperfusion is poorly understood. We studied the expression of six nuclear proto-oncogenes in porcine myocardium subjected to ischemia and reperfusion in anesthetized pigs. Stunning was achieved by two 10 min occlusions of the LAD, separated by 30 min of reperfusion. Hearts were excised after the first occlusion, after the first reperfusion and at 30, 120, 150 and 210 minutes of the second reperfusion. Total RNA was prepared from stunned as well as normally perfused myocardial tissue and subjected to Northern blotting. FosB expression was never detected. The c-myc gene was expressed, but its level was unchanged by ischemia. C-jun expression was slightly increased by ischemia (3.1*0.6-fold). C-fos, Egr-7 and the junS genes were highly induced being 5 to 7 fold. In three animals, pretreated with the Ot-antagonist metoprolol and then subjected to the experimental protocol, proto-oncogene expression wassimilar to non-blocked controls. Our results show that the myocardium responds to ischemic stress by the induction of at least four transcription factors which may be further operative in repair processes.

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Rakesh C. Kukreja, ‘Suritmiw K. Bairn, Kathryn E. foesser and Michael L. Hess, Division of Cardiology, hfedkxd College of Virginia, Richmond Va 23295, USA and Duke Univwsity h4edical Center. Durhm, NC 27710 UsA Preco&itioning d the heart by repaated stunning (brief pedoda d ocdusio& with ir&nittent rep&u&i) improves myocardii aatvage after proknged tschemia and induces transktkn of heat shock protein (HSP). Since oxygenderived free radii are known to be generated during kchemidrepeduskn (t/R) injury in the heart, we hypothesized that brief perkds d oxidattve bursta generated by ischemk preoonditkning might resuit in increased expression of HSP70 which would eventuatty protect the tissue against free radical mediied reperk&n injury, We therefore studied the direct expression Or HSP70 mRNA using human HSP70 speci5c DNA probe in Langemkrff Mated perfused rat hearts by oxygen radicals and compared thii to l/R injury. Five groups of hearts were studkd 1. Control; 2. hearts perfused with xanthine (X)(1 00 PM). 3. pedused with xanthine oxidaee (XO)(O.oS U/ml); 3. X and X0 (which generates superoxide radical and H20 ) for 15 min, 4. X and X0 in pmaence of SOD (SO U/ml) and 5. hearts subjected to SO min kchemta fottowed by 18 mm. repeduakn. Northern bkt analysis of total RNA from these tissues revealed that HSP70 mRNA was negligibk or undetectabk in controts, X or X0 perfused controi hearts. However the expression of HSP 70 was severatfok hiihar in hearts peduaed with X pius X0 and l/R groups but was kwer or undetectable when pedused in the presence of SOD. For the first time, these results demonstrate that free radicals directty increase gene expreeskn of HSP 70 whkh was comparable to hearts subjected to I/R. We conclude that one of the mechanisms of expression al HSP 70 by preconditioning are oxygen radiials. s.101