An improved isolation procedure for adult mouse cardiomyocytes.
An improved isolation procedure for adult mouse cardiomyocytes.
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DOI：
10.1007/s12013-011-9165-9
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发表时间：
2011-09
影响因子：
2.6
通讯作者：
Ingwall, Joanne S.
中科院分区：
文献类型：
--
作者：
Pinz, Ilka;Zhu, Ming;Mende, Ulrike;Ingwall, Joanne S.
分离的成年小鼠心肌细胞是心血管研究的重要工具，但制备具有挑战性。由于能量供应决定细胞功能和活力，我们比较了分离的心肌细胞与完整小鼠心脏中的总肌酸（[Cr]）和[ATP]。分离的心肌细胞遭受严重的Cr（-70%）和ATP（-53%）损失。肌细胞不能充满[铬]在5小时的孵育期在培养基中补充1 mM铬。相反，向消化缓冲液中加入20 mM Cr足以维持正常[Cr]。用5 mM的肌苷（Ino）和腺苷（Ado）补充缓冲液以防止细胞核苷的损失，部分地保护细胞免受ATP损失。为了测试维持[ATP]和[Cr]是否改善收缩功能，通过从0.5到10 Hz变化起搏速率和在5和10 Hz加入异丙肾上腺素（Iso）来激发肌细胞。所有组表现良好，高达5 Hz，显示出积极的细胞缩短频率的关系，然而，只有16%的心肌细胞分离在标准条件下能够维持起搏与ISO挑战在10 Hz。与此相反，30-50%的肌细胞与正常铬水平能够收缩和维持低舒张[Ca 2 +]。在Cr和Cr/Ino/Cr处理组中，细胞产率也得到改善（85-90%对未处理肌细胞中的70-75%杆状）。这些数据表明，分离的心肌细胞的活力和性能得到改善时，他们被保护免受严重损失的Cr和ATP在隔离，使他们成为一个更好的研究工具。
Isolated adult mouse cardiomyocytes are an important tool in cardiovascular research, but are challenging to prepare. Because the energy supply determines cell function and viability, we compared total creatine ([Cr]) and [ATP] in isolated cardiomyocytes with the intact mouse heart. Isolated myocytes suffered severe losses of Cr (−70%) and ATP (−53%). Myocytes were not able to replete [Cr] during a 5 h incubation period in medium supplemented with 1 mM Cr. In contrast, adding 20 mM Cr to the digestion buffers was sufficient to maintain normal [Cr]. Supplementing buffers with 5 mM of inosine (Ino) and adenosine (Ado) to prevent loss of cellular nucleosides partially protected against loss of ATP. To test whether maintaining [ATP] and [Cr] improves contractile function, myocytes were challenged by varying pacing rate from 0.5 to 10 Hz and by adding isoproterenol (Iso) at 5 and 10 Hz. All groups performed well up to 5 Hz, showing a positive cell shortening–frequency relationship; however, only 16% of myocytes isolated under standard conditions were able to sustain pacing with Iso challenge at 10 Hz. In contrast, 30–50% of the myocytes with normal Cr levels were able to contract and maintain low diastolic [Ca2+]. Cell yield also improved in Cr and the Cr/Ino/Ado-treated groups (85–90% vs. 70–75% rod shaped in untreated myocytes). These data suggest that viability and performance of isolated myocytes are improved when they are protected from the severe loss of Cr and ATP during the isolation, making them an even better research tool.
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影响因子：
4.3
作者：
Starling, RC;Hammer, DF;Altschuld, RA
通讯作者：
Altschuld, RA
影响因子：
5
作者：
Lim, CC;Apstein, CS;Liao, RL
通讯作者：
Liao, RL
影响因子：
20.1
作者：
LEWANDOWSKI, ED;JOHNSTON, DL;ROBERTS, R
通讯作者：
ROBERTS, R
DOI：
10.1152/ajpendo.00259.2002
发表时间：
2003-02-01
影响因子：
5.1
作者：
Boehm, E;Chan, S;Neubauer, S
通讯作者：
Neubauer, S
影响因子：
5
作者：
Jennings, RB;Sebbag, L;Reimer, KA
通讯作者：
Reimer, KA