钙释放通道稳定蛋白基因转染对心力衰竭心室肌细胞收缩功能的影响

Effects of FKBP12.6 gene transfection on the contractility of ventricular myocytes of rats with heart failure

目的探讨钙释放通道稳定蛋白FKBP12.6基因转染对心力衰竭心室肌细胞收缩功能的影响。方法建立大鼠心力衰竭模型,采用酶解分离技术分离心室肌细胞,正常对照组用Ad-GFP感染正常心室肌细胞,Ad-FKBP12.6-GFP组用携带FKBP12.6基因的重组腺病毒Ad-FKBP12.6-GFP感染心力衰竭心室肌细胞,Ad-GFP组用Ad-GFP感染心力衰竭心室肌细胞。通过RT-PCR和Western blotting技术检测转基因的表达;在局部场刺激条件下,激光共聚焦线扫描检测心肌细胞内的钙瞬变,采用激光共聚焦面扫描测量心肌细胞舒张期末和收缩期末长度,以评价心肌细胞的收缩功能。结果Ad-FKBP12.6-GFP组的FKBP12.6 mRNA和蛋白表达水平(分别为2.48±0.08,0.94±0.11)显著高于正常对照组(分别为0.96±0.12,0.48±0.07,P<0.01)、Ad-GFP组(分别为0.47±0.08,0.25±0.04,P<0.01),且正常对照组显著高于Ad-GFP组;Ad-FKBP12.6-GFP组的钙瞬变幅度(F/F0峰值,3.16±0.42)显著高于正常对照组(2.14±0.41,P<0.05)、Ad-GFP组(1.43±0.38,P<0.01),且正常对照组显著高于Ad-GFP组(P<0.05);Ad-FKBP12.6-GFP组心室肌细胞收缩分数(9.36%±1.78%)显著高于正常对照组(8.17%±3.74%,P<0.05)、Ad-GFP组(5.24%±1.25%,P<0.01),且正常对照组显著高于Ad-GFP组(P<0.01)。结论FKBP12.6基因转染改善了心力衰竭大鼠心室肌细胞的收缩功能,上调FKBP12.6的表达可能是心力衰竭治疗的一条新途径。

Objective To investigate the effects of FKBP12. 6 gene transfection on the contractility of ventricular myocytes of rats with heart failure. Methods Rat model of heart failure was established by a surgical operation of abdominal aortic stenosis. The enzymatic hydrolysis was employed to isolate the rats＇ ventricular myocytes. Mediated by adenovirus the FKBP12. 6 gene was transfected into ventrie ular myocytes.Ad-GFP infected cardiac myocytea from heart failure rats and normal rats were used as control. Western blotting and re verse transcriptase-polymerase chain reaction （RT-PCR） were used to detect the specific overexpression of FKBP12. 6. Transfected ventricular myoeytes were loaded with the membrane-permeant Ca^2 dye X-rhod-l-AM. Under the condition of field stimulation, the laser confocal microscope in line-scan and plane-scan mode was used to detect the Ca^2 transients and myocytes contraction. Results The mRNA and protein levels of FKBP12. 6 in the myocytes of rats with heart failure were significantly lower than that in normal myocytes （0. 47±0. 08 vs 0. 96±0. 12, 0. 25±0. 04 vs 0. 48±0. 07, P〈0. 01）. Adenovirus mediated FKBP12. 6 gene transfection resulted in an increase in relative FKBP12. 6 mRNA levels （2.48±0. 08 vs0. 47±0. 08; 2.48±0. 08 vs0. 96±0. 12, P〈0. 01） and an increase in relative FKBP12. 6 protein levels （0. 94±0. 11 vs 0. 25±0.04; 0. 94±0. 11 vs 0. 48±0. 07, P〈0. 01） at 48 hours after transfection compared with those of Ad-GFP myocytes and normal myocytes. The transient intracellular Ca2 concentration was significantly decreased in Ad-GFP infected myocytes of rats with heart failure than that in normal control （peak F/F0, 1.43±0. 38 vs 2.14±0. 41, P〈0. 05）. The amplitude of the fluorescence [Ca^2＋]transient recorded in the Ad-FKBP12. 6-GFP eardiomyocytes was significantly increased compared with those in Ad-GFP infected myocytes and normal control （peak F/F0, 3.16±0. 42 vs 1.43±0. 38, P〈0. 01; 3. 16±0.42 vs 2. 14±0. 41, P〈0.05）. Cardiomyocyte fractional shortening was lower in AdGFP infected cardiomyocytes than that in normal control （5. 24%±1. 250/oo vs 8. 17%±3. 74%, P〈 0.01）. However, fractional shortening was increased significantly in Ad-FKBP12. 6 GFP cardiomyocytes compared with those in Ad-GFP infectedmyocytes （9.36%±1.78% vs5.24%＋1.25%, P〈0.01; 9.36% ±1.78% vs8.17% ±3.74% , P〈0.05）. Conclusion FKBP12. 6 gene transfection may increase SR Ca^2＋ release and consequently improve the contractility of ventricular myocytes of rats with heart failure, therefore be possible as a potential therapeutic way for heart failure.