Experimental investigation of direct myocardial protective effect of atrial natriuretic peptide in cardiac surgery Shinji Wakui, MD, Akira Sezai, MD,

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Experimental investigation of direct myocardial protective effect of atrial natriuretic peptide in cardiac surgery Shinji Wakui, MD, Akira Sezai, MD, PhD, Gero Tenderich, MD, PhD, Mitsumasa Hata, MD, PhD, Syunji Osaka, Yoshiki Taniguchi, Reiner Koerfer, MD, PhD, Kazutomo Minami, MD, PhD The Journal of Thoracic and Cardiovascular Surgery Volume 139, Issue 4, Pages 918-925 (April 2010) DOI: 10.1016/j.jtcvs.2009.08.030 Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 1 Experimental method and groups. Thirty-two pigs underwent surgery with cardiopulmonary bypass (CPB) and were divided into 4 groups according to timing of atrial natriuretic peptide (ANP) administration. In group C (n = 8), there was no use of atrial natriuretic peptide after aortic (Ao) crossclamping; only cardioplegia (Miotecter; Mochida Pharmaceutical Co, Ltd, Tokyo, Japan) was used. After 30 minutes of cardioplegia, reperfusion was performed for 60 minutes. In group H1 (n = 8), atrial natriuretic peptide (100 μg) was administered after aortic crossclamping through ascending aorta. In group H2 (n = 8), atrial natriuretic peptide was administered before aortic declamping. In group H1 + H2 (n = 8), atrial natriuretic protein was administered both after aortic crossclamping and before aortic declamping. The Journal of Thoracic and Cardiovascular Surgery 2010 139, 918-925DOI: (10.1016/j.jtcvs.2009.08.030) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 2 Blood cyclic guanosine monophosphate (cGMP) concentrations. Concentrations were significantly increased in groups H1 + H2 and H2 relative to group C. ∗1 indicates P < .0001 group H1 + H2 versus C; ∗2 indicates P < .0001 group H1 + H2 versus C; ∗3 indicates P < .007 group H1 + H2 versus C; ∗4 indicates P < .0014 group H2 versus C; ∗5 indicates P < .0007 group H2 versus C; ∗6 indicates P < .003 group H2 versus C; ∗7 indicates P < .0138 group H1 + H2 versus H1; ∗8 indicates P < .0294 group H1 + H2 versus H1; ∗9 indicates P < .0424 group H1 versus H2. The Journal of Thoracic and Cardiovascular Surgery 2010 139, 918-925DOI: (10.1016/j.jtcvs.2009.08.030) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 3 Residual myocardial adenosine triphosphate (ATP) levels. No significant differences were observed among treatment groups (H1, H2, H1 + H2); however, residual adenosine triphosphate level tended to increase in groups H1, H2, and H1 + H2 relative to group C. The Journal of Thoracic and Cardiovascular Surgery 2010 139, 918-925DOI: (10.1016/j.jtcvs.2009.08.030) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 4 Myocardial cyclic guanosine monophosphate (cGMP) concentrations. Concentration was significantly increased in groups H2 and H1 + H2 relative to group C. ∗1 indicates P < .0001 H1 + H2 versus C; ∗2 indicates P < .0001 H1 + H2 versus H1; ∗3 indicates P < .0001 H2 versus C; ∗4 indicates P < .0001 H2 versus H1. wg, Wet weight in grams. The Journal of Thoracic and Cardiovascular Surgery 2010 139, 918-925DOI: (10.1016/j.jtcvs.2009.08.030) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 5 Myocardial calcium (Ca) concentration. Increase in concentration was significantly inhibited in group H1 + H2 relative to group C. ∗1 indicates P < .0038 H1 + H2 versus C. wetg, Wet weight in grams. The Journal of Thoracic and Cardiovascular Surgery 2010 139, 918-925DOI: (10.1016/j.jtcvs.2009.08.030) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 6 Electron microscopic images. A, Mitochondria in electron microscopy. Swelling of mitochondria with sparse cristae was observed in group C. In contrast, normal mitochondria were preserved and large amounts of glycogen granules were seen in human atrial natriuretic peptide (hANP) treatment groups. B, Myocardial fibers in electron microscopy. Large numbers of thick, fuzzy contraction bands were observed in group C. In contrast, normal distinct myocardial fibers were preserved in treatment groups. C, Myocardial nuclei in electron microscopy. Aggregates of nuclear chromatin or aggregation of chromatin around nuclei were markedly observed in group C; however, these changes were minor in groups, with nuclei equivalent to those in nonischemic myocardium. The Journal of Thoracic and Cardiovascular Surgery 2010 139, 918-925DOI: (10.1016/j.jtcvs.2009.08.030) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 7 Nuclear chromatin grades and ischemic changes in nuclear chromatin. Nuclear chromatin in myocardial cells were observed with light microscopy and classified according to 4 grades: normal (−), slight ischemia (+), hyperischemia (++), irreversible ischemia (+++). Ischemic changes were inhibited in treatment groups (H1 + H2 more so than H1 and H2). All 4 groups were treated with cardioplegic solution, so incidence of irreversible ischemic change was 0%. The Journal of Thoracic and Cardiovascular Surgery 2010 139, 918-925DOI: (10.1016/j.jtcvs.2009.08.030) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 8 Time course of changes in calcium (Ca) concentration ([Ca], [Ca2+]) during ischemia–reperfusion. Increase in calcium was more remarkable before reperfusion (during ischemia, 1) than immediately after reperfusion (2), contrary to our expectations. There was slight increase immediately after reperfusion, and then levels decreased. Thus myocardial calcium increase occurred mainly during ischemia. Data from Marban and associates.20 ANP, Atrial natriuretic peptide. The Journal of Thoracic and Cardiovascular Surgery 2010 139, 918-925DOI: (10.1016/j.jtcvs.2009.08.030) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions