The increasing utility of transgenic mice in molecular studies of the cardiovascular system has motivated us to characterize the ionic currents in neonatal mouse ventricular myocytes. 2. Cell capacitance measurements (30 +/- 1 pF, n = 73) confirmed visual impressions that neonatal mouse ventricular myocytes in primary culture are considerably smaller than freshly isolated adult ventricular myocytes. With the use of electron microscopy, mitochondria and sarcoplasmic reticulum were found in close association with myofibrils, but transverse tubules were not observed. 3. Action potential durations, measured at 50 and 90% repolarization, were 23 +/- 1 and 42 +/- 2 ms respectively (n = 46). Application of 4-aminopyridine (4-AP; 5 mM) prolonged action potential duration at 50% repolarization by 26 +/- 5% (n = 3). The brevity of the action potential is explained by the rapid activation of a transient outward K+ current upon voltage-clamp depolarization to plateau potentials. 4. Potassium currents identified include an inward rectifier, a large 4-AP-sensitive transient outward, a slowly inactivating 4-AP-insensitive outward, a slowly activating delayed rectifier and a small rapidly activating E-4031 (10 microM)-sensitive delayed rectifier K+ current. 5. Sodium currents (-305 +/- 50 pA pF-1, n = 21) were recorded in 40 mM Na+ with Ni2+ (1 mM) to block Ca2+ currents and with K+ replaced by Cs+. The relative insensitivity of the Na+ current to block by tetrodotoxin (IC50 = 2.2 +/- 0.3 microM, n = 4) is distinctive of the cardiac Na+ channel isoform. 6. Nitrendipine-insensitive (10 microM) Ba2+ currents elicited during steps from -90 to -30 mV measured -25 +/- 5 pA pF-1 (n = 7, 30 mM Ba2+). Decay of these currents was complete during 180 ms depolarizations, even with Ba2+ as the charge carrier. These currents were not present when the holding potential was set at -50 mV. These data support the presence of a low threshold, T-type Ca2+ current. 7. The maximal nitrendipine-sensitive L-type Ca2+ current density was -10 +/- 2 pA pF-1 (n = 8) in 2 mM Ca2+ and -38 +/- 5 pA pF-1 (n = 9) in 30 mM Ba2+. Exposure to isoprenaline (1 microM) resulted in an 82% increase (n = 3) in the amplitude of the Ba2+ currents elicited at 0 mV. 8. Neonatal mouse cardiac myocytes in primary culture possess surprisingly large inward currents given the brevity of their action potentials.(ABSTRACT TRUNCATED AT 250 WORDS)
摘要
转基因小鼠在心血管系统分子研究中的应用日益广泛，这促使我们对新生小鼠心室肌细胞的离子电流进行表征。2. 细胞电容测量结果（30±1 pF，n = 73）证实了视觉印象，即原代培养的新生小鼠心室肌细胞明显小于新鲜分离的成年心室肌细胞。通过电子显微镜观察发现，线粒体和肌浆网与肌原纤维紧密相连，但未观察到横管。3. 在复极化50%和90%时测量的动作电位持续时间分别为23±1和42±2毫秒（n = 46）。应用4-氨基吡啶（4-AP；5 mM）使复极化50%时的动作电位持续时间延长了26±5%（n = 3）。动作电位的短暂性可通过电压钳去极化至平台电位时快速激活的瞬时外向钾电流来解释。4. 确定的钾电流包括内向整流器、对4-AP敏感的大瞬时外向电流、缓慢失活的对4-AP不敏感的外向电流、缓慢激活的延迟整流器以及对E-4031（10 microM）敏感的小快速激活延迟整流器钾电流。5. 在40 mM Na+中，用Ni2+（1 mM）阻断钙电流，并用Cs+替代K+，记录到钠电流为-305±50 pA pF-1（n = 21）。钠电流对河豚毒素阻断的相对不敏感性（IC50 = 2.2±0.3 microM, n = 4）是心脏钠通道同工型的特征。6. 在从-90 mV到-30 mV的阶跃过程中引发的对尼群地平不敏感（10 microM）的Ba2+电流测量值为-25±5 pA pF-1（n = 7, 30 mM Ba2+）。即使以Ba2+作为电荷载体，在180毫秒去极化期间这些电流的衰减也已完成。当保持电位设置为-50 mV时，这些电流不存在。这些数据支持存在低阈值T型钙电流。7. 在2 mM Ca2+中，最大的对尼群地平敏感的L型钙电流密度为-10±2 pA pF-1（n = 8），在30 mM Ba2+中为-38±5 pA pF-1（n = 9）。暴露于异丙肾上腺素（1 microM）导致在0 mV时引发的Ba2+电流幅度增加82%（n = 3）。8. 考虑到其动作电位的短暂性，原代培养的新生小鼠心肌细胞具有惊人的大内向电流。（摘要截断于250字）
