Huang X D, Horackova M, Pressler M L
The Krannert Institute of Cardiology, Department of Medicine, Indiana University, Indianapolis 46202, USA.
Exp Cell Res. 1996 Nov 1;228(2):254-61. doi: 10.1006/excr.1996.0324.
In the present study, we have investigated the changes in the expression and distribution of the principal gap-junction channel protein in ventricular muscle, connexin 43 (Cx43), during the first 2 weeks of culturing adult guinea pig cardiomyocytes at low density to prevent formation of cellular contacts. In freshly isolated cardiomyocytes, immunoreactive Cx43 occupied 6.5 +/- 0.4% of the pixel area of the cell, with 85% being localized to dense particles at the step-like end projections of the myocytes (intercalated disk regions) and 15% being within the sarcoplasm or along the lateral surface of the myocytes ("nondisk" distribution). During the myocytes' first 48 h in culture, immunoreactive Cx43 decreased by 27.5% from control values, to 4.7 +/- 0.5% of the cells' pixel area (P < 0.01). Cx43 particles also redistributed: after 48 h in culture approximately 90% of the immunoreactive Cx43 was localized in the sarcoplasm and nondisk regions of the myocyte. After 7 days, immunoreactive Cx43 only occupied 50% of the cells' control pixel area (P < 0.01) and was nearly uniform in its punctate pattern throughout the sarcoplasm. This distribution remained the same during the 2nd week in culture. Changes in myosin light chain staining during 8 days in culture largely paralleled those in Cx43 staining. Laser confocal microscopic analysis of double-immunolabeled myocytes that had been in culture for 24-48 h showed colocalization of Cx43 with clathrin in approximately 30% of the sarcoplasmic Cx43 particles. Thus it is demonstrated that the expression of Cx43 decreases significantly during the first 48 h in culture after myocyte isolation and that Cx43 also undergoes substantial redistribution but for the next 2 weeks remains more or less unchanged and at relatively high levels (approximately 50%). These data indicate that cardiomyocytes in isolation maintain their ability to reconnect with each other for up to at least 2 weeks. This is the first time that this property has been investigated in cultured adult ventricular cardiomyocytes.
在本研究中，我们研究了成年豚鼠心肌细胞在低密度培养的前2周内，心室肌中主要的缝隙连接通道蛋白连接蛋白43（Cx43）的表达和分布变化，以防止细胞间接触的形成。在新鲜分离的心肌细胞中，免疫反应性Cx43占细胞像素面积的6.5±0.4%，其中85%定位于心肌细胞阶梯状末端突起（闰盘区域）的致密颗粒，15%位于肌浆内或沿着心肌细胞的侧面（“非盘状”分布）。在培养的最初48小时内，免疫反应性Cx43从对照值下降了27.5%，降至细胞像素面积的4.7±0.5%（P<0.01）。Cx43颗粒也发生了重新分布：培养48小时后，约90%的免疫反应性Cx43定位于心肌细胞的肌浆和非盘状区域。7天后，免疫反应性Cx43仅占细胞对照像素面积的50%（P<0.01），并且在整个肌浆中的点状模式几乎均匀。在培养的第2周，这种分布保持不变。培养8天期间肌球蛋白轻链染色的变化在很大程度上与Cx43染色的变化平行。对培养24 - 48小时的双免疫标记心肌细胞进行激光共聚焦显微镜分析显示，约30%的肌浆Cx43颗粒中Cx43与网格蛋白共定位。因此证明，Cx43的表达在心肌细胞分离后的培养最初48小时内显著降低，并且Cx43也经历了大量的重新分布，但在接下来的2周内或多或少保持不变且处于相对较高水平（约50%）。这些数据表明，分离的心肌细胞在至少2周内保持彼此重新连接的能力。这是首次在培养的成年心室心肌细胞中研究这种特性。
