Nonischemic myocardial acidosis adversely affects microvascular and myocardial function and triggers apoptosis during cardioplegia Kamal R. Khabbaz,

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Nonischemic myocardial acidosis adversely affects microvascular and myocardial function and triggers apoptosis during cardioplegia Kamal R. Khabbaz, MD, Jun Feng, MD, PhD, Munir Boodhwani, MD, Richard T. Clements, PhD, Cesario Bianchi, MD, PhD, Frank W. Sellke, MD The Journal of Thoracic and Cardiovascular Surgery Volume 135, Issue 1, Pages 139-146 (January 2008) DOI: 10.1016/j.jtcvs.2007.07.035 Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 1 Myocardial pH profiles from 3 separate experiment groups from pH 7.2, pH 6.5, and pH 6.2, respectively. B, Baseline value (30 minutes); CP, blood cardioplegic arrest (60 minutes); RP, reperfusion (10 minutes). Myocardial pH was measured with a Khuri glass electrode, the tip of which was inserted into the left ventricle. The Journal of Thoracic and Cardiovascular Surgery 2008 135, 139-146DOI: (10.1016/j.jtcvs.2007.07.035) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 2 A, Recovery of left ventricular developed pressure (LVDP). The LVDP was similar in both the control and the 3 treatment groups at baseline. *P < .05 versus baseline; †P < .01 versus pH 7.2. B, Simple linear regression analysis testing inversely the linear relationship between myocardial pH shifts and the recovery of LVDP. Recovery values on the y-axis were obtained 120 minutes after reperfusion. Mean myocardial pH represents the average myocardial pH during the 60 minutes of cardioplegic arrest. The Journal of Thoracic and Cardiovascular Surgery 2008 135, 139-146DOI: (10.1016/j.jtcvs.2007.07.035) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 3 Percent relaxation to increasing vasodilating agents after precontraction with U46619. A, Response to endothelium-dependent vasodilator adenosine 5′diphosphate (ADP). B, Linear regression analysis for testing the effect of the myocardial pH shifts on percent relaxation in response to ADP at 10−4 mol/L (y-axis). C, Response to endothelium-independent vasodilator sodium nitroprusside (SNP). *P < .05 versus baseline; †P < .05 versus pH 7.2; ‡P < .05 versus pH 6.5. The Journal of Thoracic and Cardiovascular Surgery 2008 135, 139-146DOI: (10.1016/j.jtcvs.2007.07.035) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 4 Immunoblots and graphs showing the protein levels and fold changes in total Bcl2 (A), p-Bcl2-70 = phospho-Bcl2-serine 70 (B), p-Bad-112 = phospho-Bad-serine 112 (C), and p-Bad-136 = phospho-Bad-serine 136 (D) from control, pH 7.2, pH 6.5, and pH 6.2 groups, respectively. The Journal of Thoracic and Cardiovascular Surgery 2008 135, 139-146DOI: (10.1016/j.jtcvs.2007.07.035) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 5 Graph showing the amount of caspase 3 activity in different groups. *P < .05 versus control; †P < .05 versus pH 7.2; #P < .01 versus pH 7.2. The Journal of Thoracic and Cardiovascular Surgery 2008 135, 139-146DOI: (10.1016/j.jtcvs.2007.07.035) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 6 TUNEL assay: A–D, Control hearts exhibiting TUNEL-negative green cells (arrow). Treated hearts showing TUNEL-positive brown cells in cardiomyocytes (arrow) and endothelial cells of a microvessel (arrow). E, Bar graph illustrating the percentage of TUNEL-positive nuclei from control hearts and hearts after cold blood cardioplegic arrest. *P < .05 versus control; †P < .01 versus pH 7.2; #P < .001 versus pH 7.2; ‡P < .01 versus pH 6.5. The Journal of Thoracic and Cardiovascular Surgery 2008 135, 139-146DOI: (10.1016/j.jtcvs.2007.07.035) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions