Inserte Javier, Garcia-Dorado David, Ruiz-Meana Marisol, Agulló Luis, Pina Pilar, Soler-Soler Jordi
Servicio de Cardiologia, Hospital Universitari Vall d'Hebron, Passeig Vall d'Hebron 119-129, Barcelona 08035, Spain.
Cardiovasc Res. 2004 Oct 1;64(1):105-14. doi: 10.1016/j.cardiores.2004.06.001.
It has been shown that sarcolemmal rupture can occur during reenergization in cardiomyocytes in which previous ischemia has induced sarcolemmal fragility by calpain-dependent hydrolysis of structural proteins. We tested the hypothesis that attenuated calpain activation contributes to the protection against reperfusion-induced cell death afforded by ischemic preconditioning (IPC), and investigated the involvement of protein kinase A (PKA) in this effect.
Calpain activity and degradation of different structural proteins were studied along with the extent of necrosis in isolated rat hearts submitted to 60 min of ischemia and 30 min of reperfusion with or without previous IPC (two cycles of 5 min ischemia-5 min reperfusion), and the ability of different treatments to mimic or blunt the effects of IPC were analyzed.
IPC accelerated ATP depletion and rigor onset during ischemia but reduced LDH release during reperfusion by 69% (P<0.001). At the end off reperfusion, calpain activity was reduced by 66% (P<0.001) in IPC, and calpain-dependent degradation of sarcolemmal proteins was attenuated. Addition of the calpain inhibitor MDL-28170 mimicked the effects of IPC on protein degradation and reduced LDH release by 48% (P<0.001). The effects of IPC on calpain, alpha-fodrin, and LDH release were blunted by the application of the PKA inhibitor H89 or alprenolol during IPC, while transient stimulation of PKA with CPT-cAMP or isoproterenol before ischemia attenuated calpain activation, alpha-fodrin degradation, and markedly reduced LDH release (P<0.001). In hearts exposed to Na(+)-free perfusion, IPC attenuated calpain activation by 67% (P<0.001) and reduced by 56% (P<0.001) LDH release associated to massive edema occurring during Na(+) readmission without modifying its magnitude.
These results are consistent with PKA-dependent attenuation of calpain-mediated degradation of structural proteins being an end-effector mechanism of the protection afforded by IPC.
研究表明，在先前缺血通过钙蛋白酶依赖性水解结构蛋白导致肌膜脆性增加的心肌细胞再灌注过程中，可发生肌膜破裂。我们检验了以下假设：钙蛋白酶激活减弱有助于缺血预处理（IPC）对再灌注诱导的细胞死亡的保护作用，并研究了蛋白激酶A（PKA）在这一效应中的作用。
研究了钙蛋白酶活性及不同结构蛋白的降解情况，以及在有或无先前IPC（两个5分钟缺血-5分钟再灌注周期）的情况下，离体大鼠心脏经历60分钟缺血和30分钟再灌注后的坏死程度，并分析了不同处理模拟或减弱IPC作用的能力。
IPC加速了缺血期间的ATP消耗和僵直的发生，但使再灌注期间的乳酸脱氢酶（LDH）释放减少了69%（P<0.001）。再灌注结束时，IPC组的钙蛋白酶活性降低了66%（P<0.001），肌膜蛋白的钙蛋白酶依赖性降解减弱。添加钙蛋白酶抑制剂MDL-28170模拟了IPC对蛋白降解的作用，使LDH释放减少了48%（P<0.001）。在IPC期间应用PKA抑制剂H89或阿普洛尔可减弱IPC对钙蛋白酶、α-血影蛋白和LDH释放的作用，而在缺血前用CPT-环磷酸腺苷（cAMP）或异丙肾上腺素短暂刺激PKA可减弱钙蛋白酶激活、α-血影蛋白降解，并显著减少LDH释放（P<0.001）。在无钠灌注的心脏中，IPC使钙蛋白酶激活减弱了67%（P<0.001），并使与再灌注时大量水肿相关的LDH释放减少了56%（P<0.001），同时不改变水肿的程度。
这些结果与PKA依赖性减弱钙蛋白酶介导的结构蛋白降解是IPC提供保护的终末效应机制一致。
