亚低温对猪复苏后心肌钙调蛋白激酶Ⅱ和自噬的影响

Effects of therapeutic hypothermia on myocardial Ca^2+/calmodulin-dependent protein kinaseⅡand autophagy after cardiopulmonary resuscitation in swine

目的探讨治疗性亚低温(therapeutic hypothermia,TH)对猪心肺复苏(cardiopulmonary resuscitation,CPR)后心肌钙调蛋白激酶Ⅱ(Ca^2+/calmodulin-dependent protein kinaseⅡ,CaMKⅡ)和细胞自噬的影响。方法国产健康雄性白猪20头,体质量33~40 kg,采用随机数字表法分为3组:假手术(sham,S)组(n=4)、CPR组(n=8)与TH组(n=8)。S组仅进行动物准备,不经历心脏骤停/复苏过程。CPR组和TH组采用电刺激诱发室颤8 min,然后心肺复苏5 min,制备猪心脏骤停复苏模型。实验猪复苏成功的标准为室上性自主心律伴平均动脉压>50 mmHg持续5 min以上。复苏成功后,TH组经体表冰毯将体温降至33℃,并维持至复苏后24 h,再以1℃/h复温5 h;S组和CPR组则利用冰毯仪全程维持正常体温。于复苏后6、12、24和30 h时,利用PiCCO法测定每搏输出量(stroke volume,SV)和全心射血分数(global ejection fraction,GEF),同时采用ELISA法检测血清心肌肌钙蛋白(cardiac troponin I,cTnI)的浓度、全自动生化分析仪检测血清肌酸激酶同工酶(creatine kinase-MB,CK-MB)的活性。于复苏后30 h处死动物,取左室心尖部心肌组织,采用Western blot法检测CaMKⅡ、微管相关轻链蛋白3Ⅱ(microtubule-associated protein light chain 3,LC3Ⅱ)和p62的蛋白表达水平。多组间比较采用单因素方差分析和Bonferroni事后检验。结果与S组相比,CPR组和TH组动物出现复苏后心功能障碍及心肌损伤,表现为SV和GEF值降低、血清cTnI浓度增加及CK-MB活性升高(均P<0.05)。但与CPR组相比,TH组动物在复苏6 h后SV和GEF值升高,在复苏12 h后血清cTnI浓度减少及CK-MB活性降低,组间比较差异有统计学意义[SV(mL):6 h为25.0±6.9和31.9±3.3,12 h为26.7±5.1和34.6±3.7,24 h为28.8±3.3和35.7±3.2,30 h为29.2±5.2和36.7±3.3;GEF(%):6 h为17.1±2.7和19.9±1.8,12 h为18.7±1.9和21.6±1.8,24 h为19.3±2.3和23.0±2.4,30 h为21.0±1.7和23.7±1.7;cTnI(pg/mL):12 h为564±51和466±56,24 h为534±38和427±60,30 h为476±55和375±46;CK-MB(U/L):12 h为803±164和652±76,24 h为693±96和557±54,30 h为633±91和480±77;均P<0.05]。组织检测分析显示,与S组相比,CPR组和TH组动物在复苏后心肌CaMKⅡ和LC3Ⅱ表达增加、p62表达减少(均P<0.05)。但与CPR组相比,TH组动物在复苏后心肌CaMKⅡ和LC3Ⅱ表达减少、p62表达增加(CaMKⅡ:0.73±0.06和0.58±0.05;LC3Ⅱ:0.69±0.09和0.50±0.07;p62:0.40±0.07和0.68±0.14;均P<0.05)。结论TH减轻复苏后心功能障碍与心肌损伤的机制可能与抑制心肌CaMKⅡ表达及细胞自噬等有关。

Objective To investigate the effects of therapeutic hypothermia(TH)on myocardial Ca^2+/calmodulin-dependent protein kinaseⅡ(CaMKⅡ)and cell autophagy after cardiopulmonary resuscitation(CPR)in swine.Methods Twenty healthy male domestic swine weighing 33-40 kg were randomly(random number)divided into 3 groups:sham group(n=4),CPR group(n=8)and TH group(n=8).Sham animals only underwent general preparation without experiencing cardiac arrest and resuscitation.The animal model was established by 8 min of electrically induced ventricular fibrillation and then 5 min CPR in the CPR and TH groups.Successful resuscitation was regarded as an organized rhythm with a mean arterial pressure of greater than 50 mmHg for 5 min or more.After successful resuscitation,body temperature was decreased to 33℃by a cooling blanket and then maintained until 24 h post-resuscitation,and followed by a rewarming at a rate of 1℃/h for 5 h in the TH group.A normal temperature was maintained by the blanket throughout the experiment in the sham and CPR groups.At 6,12,24 and 30 h after resuscitation,the values of stroke volume(SV)and global ejection fraction(GEF)were measured by PiCCO,and meanwhile the serum concentrations of cardiac troponin I(cTnI)were measured by ELISA assay and the serum activities of creatine kinase-MB(CK-MB)were evaluated by an automatic biochemical analyzer.At 30 h after resuscitation,the animals were sacrificed and left ventricular myocardium was obtained for the determination of CaMKⅡ,microtubule-associated protein light chain 3Ⅱ(LC3Ⅱ)and p62 expressions by Western blot.The variables were compared with One way analysis of variance and then the Bonferroni test among the three groups.Results Compared with the sham group,myocardial dysfunction and injury after resuscitation were observed in the CPR and TH groups,which were indicated by decreased SV and GEF and also increased cTnI concentration and CK-MB activity in serum(all P<0.05).Compared with the CPR group,the values of SV and GEF were significantly increased at 6 h after resuscitation,and serum cTnI concentration and CK-MB activity were significantly decreased starting 12 h after resuscitation in the TH group[SV(mL):25.0±6.9 vs 31.9±3.3 at 6 h,26.7±5.1 vs 34.6±3.7 at 12 h,28.8±3.3 vs 35.7±3.2 at 24 h,29.2±5.2 vs 36.7±3.3 at 30 h;GEF(%):17.1±2.7 vs 19.9±1.8 at 6 h,18.7±1.9 vs 21.6±1.8 at 12 h,19.3±2.3 vs 23.0±2.4 at 24 h,21.0±1.7 vs 23.7±1.7 at 30 h;cTnI(pg/mL):564±51 vs 466±56 at 12 h,534±38 vs 427±60 at 24 h,476±55 vs 375±46 at 30 h;CK-MB(U/L):803±164 vs 652±76 at 12 h,693±96 vs 557±54 at 24 h,633±91 vs 480±77 at 30 h,all P<0.05].Tissue detection indicated that the expression of CaMKⅡand LC3Ⅱwere increased while the expression of p62 was decreased in post-resuscitation myocardium in the CPR and TH groups compared with the sham group(all P<0.05).However,the expression of CaMKⅡand LC3Ⅱwere decreased and the expression of p62 was increased in post-resuscitation myocardium in the TH group compared to the CPR group(CaMKⅡ:0.73±0.06 vs 0.58±0.05;LC3Ⅱ:0.69±0.09 vs 0.50±0.07;p62:0.40±0.07 vs 0.68±0.14,all P<0.05).Conclusion The mechanism of TH alleviating post-resuscitation myocardial dysfunction and injury may be related to the inhibition of CaMKⅡexpression and cell autophagy.