Sodha Neel R, Clements Richard T, Bianchi Cesario, Sellke Frank W
Division of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
J Am Coll Surg. 2008 Jan;206(1):33-41. doi: 10.1016/j.jamcollsurg.2007.06.308. Epub 2007 Oct 1.
The protein kinase C (PKC) family consists of 12 isoforms, 6 of which have been found in human myocardium (PKC alpha, beta I/beta II, delta, epsilon, eta, and lambda/iota). These kinases function in regulation of contractility, ion channels, and in cellular protection or damage during ischemia-reperfusion injury. This study investigated the effects of controlled ischemia-reperfusion injury through cardioplegic arrest on PKC activity in patients undergoing cardiac surgery.
Myocardium and skeletal muscle were harvested from patients before and after cardiopulmonary bypass and cardioplegic arrest. Total PKC (n=12) was isolated and specifically, the PKC delta (n=8) and PKC epsilon (n=8) isoforms were immunoprecipitated for use in functional kinase assays. Cellular fractions (n=4) were separated by differential ultracentrifugation and analyzed by Western blotting for membrane translocation (an indirect indicator of increased activity). Immunofluorescent staining for PKC delta and PKC epsilon was performed on myocardial sections. An in vitro assay of hypoxic cardioplegic arrest followed by reoxygenation was performed using isolated cardiomyocytes, and apoptosis was assessed.
Cardioplegic arrest was associated with a 24.4% +/- 4.6% increase (p < 0.05) in total PKC activity, a 26.7% +/- 4.9% increase (p < 0.05) in PKC delta activity, and a 35.3% +/- 14% increase (p < 0.05) in PKC epsilon activity in myocardium. Cardioplegic arrest induced migration of PKC delta and epsilon to the z-line of the cardiomyocyte. Inhibition of PKC delta in the in vitro studies demonstrated a considerable reduction in apoptotic cells.
PKC delta and epsilon have previously been shown to mediate and protect, respectively, from ischemia-reperfusion injury after myocardial ischemia. Demonstration of increases in their activity after cardioplegic arrest provides support for their possible role in myocardial function after cardiac surgery. Isoform-specific modulators may be of potential therapeutic value in treating postoperative myocardial dysfunction.
蛋白激酶C（PKC）家族由12种亚型组成，其中6种已在人类心肌中发现（PKCα、βI/βII、δ、ε、η和λ/ι）。这些激酶在调节心肌收缩力、离子通道以及在缺血再灌注损伤期间的细胞保护或损伤方面发挥作用。本研究调查了心脏手术患者中通过心脏停搏进行的控制性缺血再灌注损伤对PKC活性的影响。
在体外循环和心脏停搏前后从患者身上获取心肌和骨骼肌。分离总PKC（n = 12），具体而言，免疫沉淀PKCδ（n = 8）和PKCε（n = 8）亚型用于功能性激酶测定。通过差速超速离心分离细胞组分（n = 4），并通过蛋白质印迹分析膜转位（活性增加的间接指标）。对心肌切片进行PKCδ和PKCε的免疫荧光染色。使用分离的心肌细胞进行体外缺氧心脏停搏后再氧合的测定，并评估细胞凋亡。
心脏停搏与心肌中总PKC活性增加24.4%±4.6%（p < 0.05）、PKCδ活性增加26.7%±4.9%（p < 0.05）以及PKCε活性增加35.3%±14%（p < 0.05）相关。心脏停搏诱导PKCδ和ε向心肌细胞的Z线迁移。体外研究中抑制PKCδ显示凋亡细胞显著减少。
先前已证明PKCδ和ε分别介导和保护心肌缺血后的缺血再灌注损伤。心脏停搏后其活性增加的证明为它们在心脏手术后心肌功能中的可能作用提供了支持。亚型特异性调节剂在治疗术后心肌功能障碍方面可能具有潜在的治疗价值。
