Salie Ruduwaan, Lochner Amanda, Loubser Dirk J
Biomedical Research and Innovation Platform, South African Medical Research Council, Parow Valley, Cape Town, South Africa.
Faculty of Medicine and Health Sciences, Division of Medical Physiology, University of Stellenbosch, Tygerberg, Cape Town, South Africa.
Cardiovasc Ther. 2017 Jun;35(3). doi: 10.1111/1755-5922.12252.
Exposure of the heart to 5 min global ischaemia (I) followed by 5 min reperfusion (R) (ischaemic preconditioning, IPC) or transient Beta 2-adrenergic receptor (B2-AR) stimulation with formoterol (B2PC), followed by 5 min washout before index ischaemia, elicits cardioprotection against subsequent sustained ischaemia. As the washout period during preconditioning is essential for subsequent cardioprotection, the aim of this study was to investigate the involvement of protein kinase A (PKA), reactive oxygen species (ROS), extracellular signal-regulated kinase (ERK), PKB/Akt, p38 MAPK and c-jun N-terminal kinase (JNK) during this period.
Isolated perfused rat hearts were exposed to IPC (1x5min I / 5min R) or B2PC (1x5min Formoterol / 5min R) followed by 35 min regional ischaemia and reperfusion. Inhibitors for PKA (Rp-8CPT-cAMP)(16μM), ROS (NAC)(300μM), PKB (A-6730)(2.5μM), ERKp44/p42 (PD98,059)(10μM), p38MAPK (SB239063)(1μM) or JNK (SP600125)(10μM) were administered for 5 minutes before 5 minutes global ischaemia / 5 min reperfusion (IPC) or for 5 minutes before and during administration of formoterol (B2PC) prior to regional ischaemia, reperfusion and infarct size (IS) determination. Hearts exposed to B2PC or IPC were freeze-clamped during the washout period for Western blots analysis of PKB, ERKp44/p42, p38MAPK and JNK.
The PKA blocker abolished both B2PC and IPC, while NAC significantly increased IS of IPC but not of B2PC. Western blot analysis showed that ERKp44/p42 and PKB activation during washout after B2PC compared to IPC was significantly increased. IPC compared to B2PC showed significant p38MAPK and JNKp54/p46 activation. PKB and ERK inhibition or p38MAPK and JNK inhibition during the washout period of B2PC and IPC respectively, significantly increased IS.
PKA activation before regional ischaemia is a prerequisite for cardioprotection in both B2PC and IPC. However, ROS was crucial only in IPC. Kinase activation during the washout phase of IPC and B2PC, albeit different, affords the same cardioprotective response.
使心脏经历5分钟全心缺血（I），随后5分钟再灌注（R）（缺血预处理，IPC），或用福莫特罗进行短暂β2 -肾上腺素能受体（B2 - AR）刺激（B2PC），然后在指数缺血前进行5分钟洗脱，可诱导对随后持续缺血的心脏保护作用。由于预处理期间的洗脱期对随后的心脏保护至关重要，本研究的目的是探讨在此期间蛋白激酶A（PKA）、活性氧（ROS）、细胞外信号调节激酶（ERK）、蛋白激酶B/蛋白激酶B（PKB/Akt）、p38丝裂原活化蛋白激酶（MAPK）和c - jun N端激酶（JNK）的作用。
将离体灌注大鼠心脏暴露于IPC（1×5分钟I / 5分钟R）或B2PC（1×5分钟福莫特罗 / 5分钟R），随后进行35分钟局部缺血和再灌注。在5分钟全心缺血/5分钟再灌注（IPC）前5分钟或在局部缺血、再灌注和梗死面积（IS）测定前给予PKA抑制剂（Rp - 8CPT - cAMP）（16μM）、ROS抑制剂（NAC）（300μM）、PKB抑制剂（A - 6730）（2.5μM）、ERKp44/p42抑制剂（PD98,059）（10μM）、p38MAPK抑制剂（SB239063）（1μM）或JNK抑制剂（SP600125）（10μM）5分钟，或在给予福莫特罗（B2PC）前及给药期间5分钟给予上述抑制剂。在洗脱期对暴露于B2PC或IPC的心脏进行冷冻钳夹，用于对PKB、ERKp44/p42、p38MAPK和JNK进行蛋白质印迹分析。
PKA阻滞剂消除了B2PC和IPC的保护作用，而NAC显著增加了IPC组的梗死面积，但对B2PC组无此作用。蛋白质印迹分析显示，与IPC相比，B2PC后洗脱期ERKp44/p42和PKB的活化显著增加。与B2PC相比，IPC显示出显著的p38MAPK和JNKp54/p46活化。分别在B2PC和IPC的洗脱期抑制PKB和ERK或抑制p38MAPK和JNK，显著增加了梗死面积。
局部缺血前PKA活化是B2PC和IPC心脏保护的前提条件。然而，ROS仅在IPC中起关键作用。IPC和B2PC洗脱期的激酶活化虽不同，但产生相同的心脏保护反应。
