Feng Jun, Bianchi Cesario, Li Jianyi, Sellke Frank W
Division of Cardiothoracic Surgery, Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
Ann Thorac Surg. 2004 Apr;77(4):1384-9; discussion 1389-90. doi: 10.1016/j.athoracsur.2003.09.053.
Expression of Bcl-2 family proteins and activation of terminal caspase 3 are important for ischemia-reperfusion-induced apoptosis. Bad and Bax are pro-apoptotic proteins, whereas, phosphorylation of Bad inhibits its binding to and inactivation of anti-apoptotic Bcl-2. Thus, decreases in phospho-Bad would be proapoptotic. We investigated if blood (BCP) or crystalloid cardioplegia (CCP) differentially affects apoptosis gene-related proteins.
Rabbit hearts were perfused with Krebs-Henseleit buffer (KHB) on a Langendorff apparatus. Control hearts (n = 6) were perfused for 90 minutes without cardioplegic ischemia. In the other two groups, hearts were arrested for 30 minutes (37 degrees C) with BCP (n = 6) or with CCP (n = 6) administered continuously (1.5 mL/min). The hearts were reperfused for 30 minutes with KHB. Left ventricle (LV) performance was measured before cardioplegic arrest and at 30 minutes of reperfusion. In vitro relaxation responses of precontracted microvessels (100-180 microm) were obtained in a pressurized no-flow state. Total and activated or phosphorylated caspase 3, Bcl-2, Bad, and Bax were measured by quantitative immunoblotting using specific antibodies.
Blood cardioplegia significantly improved the recovery of LV developed pressure compared to CCP (p < 0.05). The endothelium-dependent relaxation in response to adenosine 5'-diphosphate was greater after BCP than after CCP (59.9 +/- 4% vs 26.9 +/- 6%, respectively; p < 0.05). There were no differences in total protein levels of caspase 3, Bcl-2, Bad, and Bax between the groups. Both BCP and CCP increased caspase 3 activity as compared with controls, but CCP caused more activation of caspase 3 than BCP (6.2 +/- 0.7 fold vs 3.1 +/- 0.4, p < 0.05). Both BCP and CCP induced phosphorylation of Bad at Ser(112), but BCP caused greater phosphorylation of Bad (3.5 +/- 0.2 fold vs 2.0 +/- 0.12 fold, respectively, p < 0.05) than CCP.
Blood cardioplegia is superior to CCP in inhibiting the activation of caspase 3 and in increasing phospho-Bad. These actions of BCP were associated with improved LV function and endothelium-dependent relaxation of coronary microvessels. These results may provide molecular mechanisms by which to improve myocardial protection during cardiac surgery.
Bcl-2家族蛋白的表达及终末半胱天冬酶3的激活对缺血再灌注诱导的细胞凋亡很重要。Bad和Bax是促凋亡蛋白，而Bad的磷酸化会抑制其与抗凋亡蛋白Bcl-2的结合及使其失活。因此，磷酸化Bad水平降低会促进细胞凋亡。我们研究了血液停搏液（BCP）或晶体停搏液（CCP）对凋亡相关基因蛋白的影响是否存在差异。
在Langendorff装置上用Krebs-Henseleit缓冲液（KHB）灌注兔心脏。对照组心脏（n = 6）在无停搏液缺血的情况下灌注90分钟。在另外两组中，心脏在37℃下用BCP（n = 6）或CCP（n = 6）持续灌注（1.5 mL/min）30分钟以使其停搏。心脏再用KHB灌注30分钟。在停搏前及再灌注30分钟时测量左心室（LV）功能。在加压无血流状态下获得预收缩微血管（100 - 180微米）的体外舒张反应。使用特异性抗体通过定量免疫印迹法测量总半胱天冬酶3、活化或磷酸化的半胱天冬酶3、Bcl-2、Bad和Bax。
与CCP相比，血液停搏液能显著改善左心室舒张末压的恢复（p < 0.05）。BCP后对5'-二磷酸腺苷的内皮依赖性舒张反应大于CCP（分别为59.9 ± 4% 对26.9 ± 6%；p < 0.05）。各组间半胱天冬酶3、Bcl-2、Bad和Bax的总蛋白水平无差异。与对照组相比，BCP和CCP均增加了半胱天冬酶3的活性，但CCP导致的半胱天冬酶3激活程度高于BCP（6.2 ± 0.7倍对3.1 ± 0.4倍，p < 0.05）。BCP和CCP均诱导Bad在Ser(112)位点的磷酸化，但BCP导致的Bad磷酸化程度高于CCP（分别为3.5 ± 0.2倍对2.0 ± 0.12倍，p < 0.05）。
血液停搏液在抑制半胱天冬酶3激活及增加磷酸化Bad方面优于CCP。BCP的这些作用与左心室功能改善及冠状动脉微血管的内皮依赖性舒张有关。这些结果可能为改善心脏手术期间心肌保护提供分子机制。
