细胞骨架对肥大心肌细胞生物力学特性的作用研究

Biomechanical Variation of Hypertrophic Cardiomyocytes and Its Correlation with the Cytoskeleton

目的观察肥大心肌细胞骨架、生物力学特性的变化规律,并进一步阐明细胞骨架改变与肥大心肌细胞生物力学特性的相关性。方法将肾上腺素加入新生大鼠心肌细胞培养后,通过微管吸吮法检测心肌细胞黏弹性;心肌细胞收缩力以细胞收缩前后表面积的变化表示。分别采用免疫细胞荧光化学与Western Blot检测细胞骨架蛋白的分布、密度及含量;通过Western Blot检测了细胞骨架蛋白磷酸化水平及细胞骨架相关蛋白激酶的活性。结果与正常心肌细胞比较,肥大心肌细胞的收缩性仅轻度升高,其弹性下降,黏性增加(P<0.01)。肥大心肌细胞骨架成分蛋白含量较正常心肌细胞明显增加(P<0.01)。与正常心肌细胞比较,肥大心肌细胞骨架蛋白actin、α-actinin、α-tubulin及desmin磷酸化水平明显升高(P<0.01)。肥大心肌细胞内PTK活性较正常心肌细胞显著增高(P<0.01)。结论研究结果表明在致心肌肥大的病理条件下,心肌细胞被动生物力学特性(黏弹性)的改变较其收缩性的变化更为显著;心肌细胞骨架蛋白的改变,尤其骨架蛋白的含量及磷酸化水平方面的变化可能对肥大心肌细胞生物力学特性的调节起到了关键性的作用。

Objective To investigate the regulative role of the cytoskeleton on the biomechanics（both initiative contractibility and the passive biomechanic properties） of hypertrophic cardiomyocytes and the possi- ble machnism. Methods The rat hypertrophic cardiomyocytes were incorporated with adnephrin. Cardiomyocytes contractile amplitude was figured by the cell area changes before and after cell contraction. Viscoelastic biomechanic properties of the cardiomyocytes were measured by micropipette aspiration technique. The distribution, density and content changes of eytoskeletal proteins were observed respectively by immunocytochemistry technique and Western Blot. The phosphorylation level of the myocardial cytoskeletal protein was detected by immunoprecipitation. The activity of cytoskeletal relative protein kinase was measured. Results In comparison with normal contrast, the contractibility and beating frequency of the hypertrophic cardiomyocytes increased slightly; the viscous coefficient of the hypertrophic cardiomyocytes increased markedly while the elastic coefficient decreased obviously（P 〈 0. 01）. The density and content of the hypertrophic myocardial eytoskeletal protein were much more than the normal ones（ P 〈 0. 01）. The distribution of hypertrophic myocardial cytoskeletal protein was irregular and alignment was indiscriminate. In contrast with the normal group, the phosphorylation level of the myocardial cytoskeletal protein and the activity of the cytoskeletal relative protein kinase in the hypertrophic cardiomyocytes increased markedly（ P 〈 0. 01）. Conclusion Analysis of the result strongly revealed that the change of passive biomechanic property of cardiomyocytes should be more important than that of the contractibility on the condition of the cardiac hypertrophic pathology. Moreover, the change of the cytoskeletal protein, in particular their distribution, density and phosphorylation level may play an important role in both the initiative contractibility and passive biomechanic properties of the hypertrophic cardiomyocytes.