Myocyte contractility can be maintained by storing cells with the myosin ATPase inhibitor 2,3 butanedione monoxime.
Myocyte contractility can be maintained by storing cells with the myosin ATPase inhibitor 2,3 butanedione monoxime.
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DOI：
10.14814/phy2.12445
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发表时间：
2015-06
影响因子：
2.5
通讯作者：
Campbell KS
中科院分区：
文献类型：
--
作者：
Chung CS;Mechas C;Campbell KS
分离的完整肌细胞可用于研究收缩机制和筛选新的治疗化合物。这些实验通常需要对动物实施安乐死，每天分离新鲜细胞，或分析培养的肌细胞，这些细胞很快就会失去杆状形态。最近的数据表明，使用低温和N-苄基-对甲苯磺酰胺（骨骼肌球蛋白ATP酶抑制剂）可以延长犬肌细胞的活力。我们在大鼠肌细胞中进行了类似的研究，以测试心肌肌球蛋白ATP酶抑制剂2，3-丁二酮单肟（BDM）和blebbistatin是否有助于维持细胞水平的功能多天。从大鼠中分离肌细胞，并将其分批储存在4°C下的HEPES缓冲液中，该缓冲液含有0.5 mmol L−1 Ca 2+和（1）无肌球蛋白ATP酶抑制剂;（2）10 mmol L−1 BDM;或（3）3 μmol L−1 blebbistatin。在分离后3天内，通过测量钙瞬变和无钙台氏液中电刺激诱导的无负荷缩短曲线来评估肌细胞的功能活力。在没有肌球蛋白ATP酶抑制剂的情况下储存的细胞具有改变的形态（更少的杆状细胞、更短的舒张期肌节长度和膜起泡），并且在24小时后不能用于收缩试验。细胞在BDM中保存48 h后仍能保持形态和收缩功能。储存在blebbistatin保持细胞形态72小时，但抑制收缩。这些数据表明，用肌球蛋白ATP酶抑制剂储存细胞可以延长将用于功能测定的肌细胞的活力。这可能有助于改进和减少在实验中使用动物。
Isolated intact myocytes can be used to investigate contractile mechanisms and to screen new therapeutic compounds. These experiments typically require euthanizing an animal and isolating fresh cells each day or analyzing cultured myocytes, which quickly lose their rod-shaped morphology. Recent data suggest that the viability of canine myocytes can be prolonged using low temperature and N-benzyl-p-toluene sulfonamide (an inhibitor of skeletal myosin ATPase). We performed similar studies in rat myocytes in order to test whether the cardiac myosin ATPase inhibitors 2,3-Butanedione monoxime (BDM) and blebbistatin help to maintain cell-level function over multiple days. Myocytes were isolated from rats and separated into batches that were stored at 4°C in a HEPES-buffered solution that contained 0.5 mmol L−1 Ca2+ and (1) no myosin ATPase inhibitors; (2) 10 mmol L−1 BDM; or (3) 3 μmol L−1 blebbistatin. Functional viability of myocytes was assessed up to 3 days after the isolation by measuring calcium transients and unloaded shortening profiles induced by electrical stimuli in inhibitor-free Tyrode's solution. Cells stored without myosin ATPase inhibitors had altered morphology (fewer rod-shaped cells, shorter diastolic sarcomere lengths, and membrane blebbing) and were not viable for contractile assays after 24 h. Cells stored in BDM maintained morphology and contractile function for 48 h. Storage in blebbistatin maintained cell morphology for 72 h but inhibited contractility. These data show that storing cells with myosin ATPase inhibitors can extend the viability of myocytes that will be used for functional assays. This may help to refine and reduce the use of animals in experiments.