单膜片钳大鼠心肌细胞中的强直张力：肌原纤维肌酸激酶的作用

Rigor tension in single skinned rat cardiac cell: role of myofibrillar creatine kinase.

作者信息

Veksler V I, Lechene P, Matrougui K, Ventura-Clapier R

机构信息

Laboratoire de Cardiologie Cellulaire et Moléculaire INSERM U-446, Faculté de Pharmacie, Université Paris-Sud, Châtenay-Malabry, France.

出版信息

Cardiovasc Res. 1997 Dec;36(3):354-62. doi: 10.1016/s0008-6363(97)00178-8.

DOI:10.1016/s0008-6363(97)00178-8

PMID:9534856

Abstract

OBJECTIVE

To elucidate the role of bound creatine kinase in adenine nucleotide compartmentation in myofibrils, the effects of this enzyme's substrates and products on rigor tension were studied in using isolated skinned rat cardiomyocytes rather than fibers, to avoid restrictions due to concentration gradients within the multicellular preparations.

METHODS

A new experimental set-up was built to allow continuous and stable measurements of force developed by cells. Triton X-100-treated cardiomyocytes were glued between a glass holder and the needle of a galvanometer. A feedback system allowed the precise measurement of force by recording the coil current necessary to prevent movement of the needle.

RESULTS

At very low [Ca2+] (pCa 7), as MgATP level decreased, rigor tension appeared. In the absence of phosphocreatine (PCr), this tension started to rise at MgATP concentrations several times higher than in the presence of 12 mM PCr. In the absence of PCr, the pMgATP/tension curves of single cells usually had a complicated relationship which could not be analyzed by a simple Hill equation. In the absence of PCr, 250 microM MgADP strongly potentiated rigor tension development in the 1 mM-3 microM range of [MgATP]; at 100 microM MgATP, in the presence of MgADP, the tension was 4.6 times higher than in the absence of MgADP. Addition of 12 mM PCr immediately eliminated rigor. Finally, in the presence of 100 microM MgATP and 250 microM MgADP, a decrease in PCr resulted in rigor; the half-maximal contracture being recorded at 1 mM PCr.

CONCLUSIONS

These results indicate a myofibrillar compartmentation of adenine nucleotides influenced by bound creatine kinase, since at equal MgATP concentrations in extramyofibrillar milieu the response of myofibrils strongly depends on the presence of PCr. Local accumulation of ADP in myofibrils due to a fall in cellular PCr and inability of myofibrillar creatine kinase to rephosphorylate ADP produced by myosin ATPase could be an important mechanism of diastolic tension rise in ischaemic conditions.

摘要

目的

为阐明结合型肌酸激酶在肌原纤维中腺嘌呤核苷酸分隔中的作用，本研究使用分离的去表皮大鼠心肌细胞而非肌纤维，研究该酶的底物和产物对强直张力的影响，以避免因多细胞制剂内浓度梯度造成的限制。

方法

构建了一种新的实验装置，以实现对细胞产生的力进行连续稳定的测量。用 Triton X - 100处理的心肌细胞粘贴在玻璃固定器和检流计的针之间。反馈系统通过记录防止针移动所需的线圈电流来精确测量力。

结果

在极低的[Ca2+]（pCa 7）时，随着 MgATP 水平降低，出现强直张力。在没有磷酸肌酸（PCr）的情况下，这种张力在 MgATP 浓度比存在 12 mM PCr 时高几倍时开始升高。在没有 PCr 的情况下，单细胞的 pMgATP/张力曲线通常具有复杂的关系，无法用简单的希尔方程进行分析。在没有 PCr 的情况下，250 μM MgADP 在[MgATP]为 1 mM - 3 μM 范围内强烈增强强直张力的发展；在 100 μM MgATP 时，在有 MgADP 的情况下，张力比没有 MgADP 时高 4.6 倍。添加 12 mM PCr 可立即消除强直。最后，在存在 100 μM MgATP 和 250 μM MgADP 的情况下，PCr 的减少导致强直；在 1 mM PCr 时记录到半最大挛缩。

结论

这些结果表明腺嘌呤核苷酸在肌原纤维中的分隔受结合型肌酸激酶影响，因为在肌原纤维外环境中 MgATP 浓度相等时，肌原纤维的反应强烈依赖于 PCr 的存在。由于细胞内 PCr 下降导致肌原纤维中 ADP 的局部积累，以及肌原纤维肌酸激酶无法将肌球蛋白 ATP 酶产生的 ADP 重新磷酸化，可能是缺血条件下舒张期张力升高的重要机制。