Zatta Amanda J, Kin Hajime, Lee George, Wang Ningping, Jiang Rong, Lust Robert, Reeves James G, Mykytenko James, Guyton Robert A, Zhao Zhi-Qing, Vinten-Johansen Jakob
Department of Cardiothoracic Surgery, Carlyle Fraser Heart Centre/Crawford Long Hospital, Emory University School of Medicine, Atlanta, GA 30308-2225, United States.
Cardiovasc Res. 2006 May 1;70(2):315-24. doi: 10.1016/j.cardiores.2005.11.030. Epub 2006 Jan 27.
Using non-selective and selective protein kinase C (PKC) epsilon and delta isoform inhibitors, we tested the hypothesis that the cardioprotective phenotype invoked by postconditioning (postcon) is dependent on PKC signalling. Furthermore, we determined whether postconditioning alters pPKCepsilon and/or pPKCdelta in cytosolic and mitochondrial fractions.
Male Sprague-Dawley rats underwent 30 min left coronary artery (LCA) occlusion followed by 3 h of reperfusion. Rats were randomised to the following groups: Untreated, no intervention either before or after LCA occlusion; Postcon, 3 cycles of 10-s full reperfusion and 10-s re-occlusion, initiated immediately at the onset of reperfusion; Chelerythrine (non-selective PKC inhibitor, 5 mg/kg)+/-postcon; Rottlerin (PKCdelta inhibitor, 0.3 mg/kg)+/-postcon; KIE1-1 (PKCepsilon inhibitor, 3.8 mg/kg)+/-postcon. A subset of rats was employed to assess pPKCepsilon and/or pPKCdelta in sham, Isch/RP (30-min LCA occlusion followed by 30-min reperfusion), and postcon-treated hearts.
Infarct size, expressed as area of necrosis as a percentage of the area at risk, AN/AAR (%), was significantly reduced by postcon compared to control (untreated) rats (39+/-2% vs. 53+/-1% in control, P<0.001). Treatment with chelerythrine alone or the PKCepsilon antagonist KIE1-1 alone at reperfusion had no effect on infarct size compared to control. In contrast, the infarct-sparing effect of postcon was abrogated by non-selective PKC inhibition and PKCepsilon antagonism (50+/-2% and 50+/-1%, respectively, P<0.002). Inhibition of PKCdelta reduced infarct size to values comparable to that in postcon group (36+/-3% vs. 39+/-2%). However, postcon in the presence of PKCdelta inhibitor did not enhance the infarct-sparing effects (38+/-2%). In addition, pPKCepsilon in postcon hearts was significantly higher in the total cell homogenate (10338+/-1627 vs. 4165+/-608 in Isch/RP, arbitrary units), and pPKCdelta translocation to mitochondria was significantly less (>2-fold decrease) compared to Isch/RP.
These data suggest that postcon modulates PKC during early reperfusion by increasing PKCepsilon expression and translocation to a site other than the outer mitochondrial membrane, and limits translocation of PKCdelta to mitochondria and associated deleterious signalling.
使用非选择性和选择性蛋白激酶C（PKC）ε和δ亚型抑制剂，我们检验了如下假设：后适应（postcon）所引发的心脏保护表型依赖于PKC信号传导。此外，我们确定后适应是否会改变细胞溶质和线粒体组分中的磷酸化PKCε和/或磷酸化PKCδ。
雄性Sprague-Dawley大鼠经历30分钟左冠状动脉（LCA）闭塞，随后再灌注3小时。大鼠被随机分为以下几组：未处理组，LCA闭塞前后均无干预；后适应组，在再灌注开始时立即进行3个周期的10秒完全再灌注和10秒再闭塞；白屈菜红碱（非选择性PKC抑制剂，5mg/kg）+/-后适应；rottlerin（PKCδ抑制剂，0.3mg/kg）+/-后适应；KIE1-1（PKCε抑制剂，3.8mg/kg）+/-后适应。使用一部分大鼠评估假手术组、缺血/再灌注组（30分钟LCA闭塞，随后30分钟再灌注）和后适应处理心脏中的磷酸化PKCε和/或磷酸化PKCδ。
梗死面积以坏死面积占危险面积的百分比表示，即AN/AAR（%），与对照（未处理）大鼠相比，后适应显著降低了梗死面积（对照为53±1%，后适应为39±2%，P<0.001）。与对照相比，再灌注时单独使用白屈菜红碱或单独使用PKCε拮抗剂KIE1-1对梗死面积没有影响。相反，非选择性PKC抑制和PKCε拮抗作用消除了后适应的梗死面积缩小效应（分别为50±2%和50±1%，P<0.002）。抑制PKCδ可将梗死面积降低至与后适应组相当的值（36±..
