Bénardeau A, Hatem S N, Rücker-Martin C, Tessier S, Dinanian S, Samuel J L, Coraboeuf E, Mercadier J J
Laboratoire de Cardiologie Moléculaire et Cellulaire, Université de Paris XI-CNRS URA 1159, Hôpital Marie Lannelongue, France.
J Mol Cell Cardiol. 1997 May;29(5):1307-20. doi: 10.1006/jmcc.1996.0366.
We examined changes in the structural and physiological characteristics of human atrial myocytes during primary culture in the presence of serum. Action potentials and ionic currents were recorded in freshly dissociated (FM) and cultured (CM) whole-cell patch-clamped myocytes, alpha-smooth muscle actin, sarcomeric alpha-actinin and beta-myosin heavy chains (beta-MHC) were stained with monoclonal antibodies. From day 5 to day 21, myocytes lost their rod shape, spread and exhibited reorganized sarcomeres. These morphological changes were associated with a marked increase in membrane capacitance (+266%). Both beta-MHC and alpha-smooth muscle actin were expressed in CM but not in FM, indicating a dedifferentiation process. CM were characterized by a lower resting potential (-30 +/- 2 v -60 +/- 4 mV, P < 0.05) and, when repolarized, by a shorter action potential duration (APD) than FM (APD-60: 126.9 v 159.6 ms, P < 0.05). The inward rectifier K+ current was absent in CM, thus explaining the low resting potential. The density of the transient component of the voltage-activated K+ current Ito1 was not modified during culture, while that of the sustained component Isus was increased fourfold. The amplitude of ICa was increased, but its density was unchanged, indicating that CM maintained a normal density of functional calcium channels. Neither the voltage dependence nor the inactivation of ICa was modified in CM. The time constants of inactivation of ICa were unchanged, although the amplitude of the rapidly inactivating component of ICa was increased in CM compared to FM. Moreover, ICa was increased by the beta-adrenergic agonist isoproterenol (1 microM) throughout the culture period. Our results demonstrate that in long-term serum-supplemented culture, adaptation of human atrial myocytes to their new environment is associated with differential alterations of the main ionic currents and phenotypic changes characteristic of immature myocardium.
我们研究了在有血清存在的原代培养过程中人心房肌细胞结构和生理特征的变化。在新鲜解离的（FM）和培养的（CM）全细胞膜片钳记录的肌细胞中记录动作电位和离子电流，用单克隆抗体对α-平滑肌肌动蛋白、肌节α-辅肌动蛋白和β-肌球蛋白重链（β-MHC）进行染色。从第5天到第21天，肌细胞失去杆状形态，铺展并呈现肌节重组。这些形态学变化与膜电容显著增加（+266%）相关。β-MHC和α-平滑肌肌动蛋白在CM中表达，但在FM中不表达，表明存在去分化过程。CM的特征是静息电位较低（-30±2V对-60±4mV，P<0.05），复极化时动作电位时程（APD）比FM短（APD-60：126.9对159.6ms，P<0.05）。CM中不存在内向整流钾电流，从而解释了低静息电位。电压激活钾电流Ito1的瞬态成分密度在培养过程中未改变，而持续成分Isus的密度增加了四倍。ICa的幅度增加，但其密度未改变，表明CM维持了功能性钙通道的正常密度。CM中ICa的电压依赖性和失活均未改变。ICa失活的时间常数未改变，尽管与FM相比，CM中ICa快速失活成分的幅度增加。此外，在整个培养期间，β-肾上腺素能激动剂异丙肾上腺素（1μM）使ICa增加。我们的结果表明，在长期补充血清的培养中，人心房肌细胞对新环境的适应与主要离子电流的差异改变以及未成熟心肌特征性的表型变化有关。
