Hongo K, Kusakari Y, Konishi M, Kurihara S, Mochizuki S
Department of Internal Medicine IV, The Jikei University, School of Medicine, 3-25-8 Nishishinbashi, Minato-ku, Tokyo 105-8461, Japan.
Pflugers Arch. 1998 Oct;436(5):639-45. doi: 10.1007/s004240050683.
To estimate myofibrillar responsiveness to Ca2+, we used the relation between cell length and intracellular [Ca2+] ([Ca2+]i) during tetanic contractions of isolated ventricular myocytes. Enzymatically isolated rat ventricular myocytes were loaded with fura-2 AM (4 microM for 10 min) and excited alternately at 340 nm and 380 nm. The ratio (R) of fura-2 fluorescence at these wavelengths [F(340)/F(380), an index of [Ca2+]i] and cell length (L) were measured simultaneously. Following treatment with thapsigargin (0.2 microM), myocytes were stimulated at 10 Hz for 10 s to produce a tetanic contraction every min and an instantaneous plot of R vs L (R-L trajectory) was constructed. The R-L trajectory followed the same path during cell shortening and re-lengthening, suggesting that dynamic equilibrium between R and L was achieved during tetanus. Increasing the extracellular [Ca2+] from 1 to 8 mM extended the R-L trajectory without a substantial shift of the relation. The Ca2+-sensitizing thiadiazinone derivative, EMD57033 (1 microM), shifted the R-L trajectory to the left (sensitization of the myofibrils to Ca2+), whereas the non-selective phosphodiesterase inhibitor, 3-isobutyl-1-methylxantine (200 microM), shifted the R-L trajectory to the right (desensitization of the myofibrils to Ca2+), in agreement with previous results obtained using skinned preparations. We conclude that the R-L trajectory is useful for estimating the myofibrillar responsiveness to Ca2+ in isolated myocytes and may be beneficial for the evaluation of inotropic agents.
为了评估肌原纤维对Ca2+的反应性，我们利用了分离的心室肌细胞强直收缩期间细胞长度与细胞内[Ca2+]（[Ca2+]i）之间的关系。用酶法分离大鼠心室肌细胞，并用fura-2 AM（4 microM，处理10分钟）加载，然后在340 nm和380 nm交替激发。同时测量这两个波长下fura-2荧光的比率（R）[F(340)/F(380)，[Ca2+]i的一个指标]和细胞长度（L）。在用毒胡萝卜素（0.2 microM）处理后，每分钟以10 Hz刺激肌细胞10秒以产生强直收缩，并构建R与L的即时图（R-L轨迹）。在细胞缩短和重新延长过程中，R-L轨迹遵循相同的路径，这表明在强直收缩期间R和L之间达到了动态平衡。将细胞外[Ca2+]从1 mM增加到8 mM可延长R-L轨迹，而关系曲线无实质性偏移。Ca2+敏感的噻二嗪酮衍生物EMD57033（1 microM）使R-L轨迹向左移动（肌原纤维对Ca2+的敏化），而非选择性磷酸二酯酶抑制剂3-异丁基-1-甲基黄嘌呤（200 microM）使R-L轨迹向右移动（肌原纤维对Ca2+的脱敏），这与之前使用透皮制剂获得的结果一致。我们得出结论，R-L轨迹可用于评估分离的肌细胞中肌原纤维对Ca2+的反应性，可能有助于评估正性肌力药物。
