Maekawa Naoya, Abe Jun-ichi, Shishido Tetsuro, Itoh Seigo, Ding Bo, Sharma Virendra K, Sheu Shey-Shing, Blaxall Burns C, Berk Bradford C
Cardiovascular Research Institute, Department of Medicine, University of Rochester, Rochester, NY, USA.
Circulation. 2006 May 30;113(21):2516-23. doi: 10.1161/CIRCULATIONAHA.105.563486. Epub 2006 May 22.
Pharmacological and genetic studies indicate that the (Na+)-H+ exchanger isoform 1 (NHE1) plays a critical role in myocardial ischemia and reperfusion (I/R) injury. We found that p90 ribosomal S6 kinase (RSK) phosphorylated serine 703 of NHE1, stimulating 14-3-3 binding and NHE1 activity. Therefore, we hypothesized that inhibiting RSK in cardiomyocytes would prevent NHE1 activation and decrease I/R-mediated injury.
To examine the role of RSK in vivo, we generated transgenic mice with cardiac-specific overexpression of dominant negative RSK (DN-RSK-TG). DN-RSK-TG hearts demonstrated normal basal cardiac function and morphology. However, myocardial infarction (left coronary artery occlusion for 45 minutes) in DN-RSK-TG hearts was significantly reduced at 24 hours of reperfusion from 46.9+/-5.6% area at risk in nontransgenic littermate controls to 26.0+/-4.2% in DN-RSK-TG (P<0.01). Cardiomyocyte apoptosis was significantly reduced after I/R in DN-RSK (0.9+/-0.2%) compared with nontransgenic littermate controls (6.2+/-2.6%). Importantly, activation of RSK and interaction of 14-3-3 with NHE1, necessary for agonist-stimulated NHE1 activity, were increased by I/R and inhibited by 70% in DN-RSK-TG (P<0.01). Next, we transduced rat neonatal cardiomyocytes with adenovirus-expressing DN-RSK (Ad.DN-RSK) and measured NHE1 activity. The baseline rate of pH recovery in acid-loaded cells was equal in cells expressing LacZ or DN-RSK. However, NHE1 activation by 100 micromol/L H2O2 was significantly inhibited in cells expressing DN-RSK (0.16+/-0.02 pH units/min) compared with Ad.LacZ (0.49+/-0.13 pH units/min). Apoptosis induced by 12 hours of anoxia followed by 24 hours' reoxygenation was significantly reduced in cells expressing Ad.DN-RSK (18.6+/-2.0%) compared with Ad.LacZ (29.3+/-5.4%).
In summary, RSK is a novel regulator of cardiac NHE1 activity by phosphorylating NHE1 serine 703 and a new pathological mediator of I/R injury in the heart.
药理学和遗传学研究表明，（钠）-氢交换体1型（NHE1）在心肌缺血再灌注（I/R）损伤中起关键作用。我们发现p90核糖体S6激酶（RSK）使NHE1的丝氨酸703磷酸化，刺激14-3-3结合并增强NHE1活性。因此，我们推测抑制心肌细胞中的RSK可防止NHE1激活并减少I/R介导的损伤。
为了研究RSK在体内的作用，我们构建了心脏特异性过表达显性负性RSK（DN-RSK-TG）的转基因小鼠。DN-RSK-TG小鼠的心脏表现出正常的基础心脏功能和形态。然而，在再灌注24小时时，DN-RSK-TG小鼠心脏的心肌梗死（左冠状动脉闭塞45分钟）明显减少，从非转基因同窝对照小鼠的危险区域面积的46.9±5.6%降至DN-RSK-TG小鼠的26.0±4.2%（P<0.01）。与非转基因同窝对照小鼠（6.2±2.6%）相比，DN-RSK小鼠在I/R后心肌细胞凋亡明显减少（0.9±0.2%）。重要的是，I/R可增加RSK的激活以及14-3-3与NHE1的相互作用（这是激动剂刺激NHE1活性所必需的），而在DN-RSK-TG小鼠中这种作用被抑制了70%（P<0.01）。接下来，我们用表达DN-RSK的腺病毒（Ad.DN-RSK）转导大鼠新生心肌细胞并测量NHE1活性。在表达LacZ或DN-RSK的细胞中，酸负荷细胞的pH恢复基线速率相同。然而，与Ad.LacZ（0.49±0.13 pH单位/分钟）相比，在表达DN-RSK的细胞中，100 μmol/L H2O2对NHE1的激活作用明显受到抑制（0.16±0.02 pH单位/分钟）。与Ad.LacZ（29.3±5.4%）相比，在表达Ad.DN-RSK的细胞中，缺氧12小时后再复氧24小时诱导的细胞凋亡明显减少（18.6±2.0%）。
总之，RSK是通过使NHE1丝氨酸703磷酸化来调节心脏NHE1活性的新型调节因子，也是心脏I/R损伤的新的病理介质。
