钙蛋白酶对房颤犬心房结构及功能的影响

Effect of calpain 1 on structural remodeling and contractile dysfunction in atrial fibrillation:experiment with dogs

目的观察钙蛋白酶在心房颤动(房颤)心房结构重构和收缩功能失调中的作用。方法健康杂种犬,假手术组、对照组、抑制剂组各5只,右心耳起搏600次/min 持续3周。静脉给予钙蛋白酶抑制剂(ALLM)。免疫组织化学技术观察钙蛋白酶1蛋白表达,荧光光度法测定钙蛋白酶活性;HE 染色观察肌溶解程度,Western 印迹技术测定心房肌肌钙蛋白Ⅰ(Tn Ⅰ)、肌球蛋白含量。超声测定左心房容积及功能变化。结果 3周后,对照组左心房肌溶解比率为(72±10)%,抑制剂组为(12±16)%,差异有统计学意义(P<0.01)。钙蛋白酶活性与肌溶解比率呈高度正相关(r_s=0.90961,P<0.01)。抑制剂组钙蛋白酶蛋白表达(62%±12%)低于对照组(79%±18%,P<0.01)。Tn Ⅰ、肌球蛋白含量在钙蛋白酶抑制剂组高于对照组(P<0.01)。抑制剂组左心房容积及功能的变化比对照组有显著改善。结论钙蛋白酶1活性和表达增高参与了房颤犬心房肌的结构重构和收缩功能失调,为房颤结构重构机制的研究提供了新的实验依据。

Objective To test the causal relationship between calpain activation and atrial structural changes during atrial fibrillation （AF）. Methods The tip of a spiral mono-polar pacing lead was fixed to the right atrial appendages of 15 dogs randomly divided into 3 equal groups： calpain inhibitor group, undergoing continuous pacing with the impulse of 600 beats/min for 3 weeks and intravenous injection of Nacetyl-Leu-Leu-Met （ALLM） ,a calpain inhibitor for 3 weeks ; control group, undergoing continuous pacing and intravenous injection of dimethyl sulfoxide （ DMSO; and sham operation group, given DMSO injection without pacing. Ultrasonography was used to observe the changes of the structures of left atrium and left atrial appendage and the heart function as well. Specimens of atrial muscles were obtained. Calpain 1 activity was detected by Suc-Leu-Leu-Val-Tyr-7-amino-4-methyl-coumarin method. HE staining was conducted to observe the myolysis. Western blotting was used to detect the protein expression of troponin Ⅰ （Tn Ⅰ ） and myosin. Results The left atrial ejection fraction （LAEF） of the ALLM group was （41 ±6）%, significantly higher than that of the control group [ （ 34 ± 9 ） %, P 〈 0. 05 ]. The left atrial appendage ejection fraction （LAAEF） of the ALLM group was （41 ±6）%, significantly higher than that of the control group [ （35 ± 6）%, P〈0.05]. Myolysis was extensive in the control group [ （71.5 ± 10.2）% ], relatively rare in the ALLM group [ （12.3 ± 16. 5）% ], and was not seen in the sham operation group, with significantly differences among the 3 groups （ all P 〈 0.01 ）. The calpain 1 activity was positively correlated with the degree of myolysis （ rs = 0.90 961, P 〈 0.01 ）. The Tn I level of the control group was （43 ± 12 ） % that of the sham operation group （P=0.001）, the Tn I level of the ALLM group was （51 ± 11）% that of the sham operation group （ P = 0. 002 ） and was significant higher than that of the control group （ P = 0.01 ）. The level of myosin of the control group was （51 ± 11 ） % that of the sham operation group （ P = 0. 002 ） , and that of the ALLM group was （ 149 ± 33 ） % that of the control group （P = 0.005）. Conclusion Activation of and upregulation of expression of calpain participate in the structural remodeling of left atrial cardiac muscle and contractile dysfunction. Calpain inhibitor suppresses the increased calpain activity and reverses