Kwak B R, van Kempen M J, Théveniau-Ruissy M, Gros D B, Jongsma H J
Department of Medical Physiology and Sports Medicine, Utrecht University, The Netherlands.
Cardiovasc Res. 1999 Nov;44(2):370-80. doi: 10.1016/s0008-6363(99)00196-0.
Primary cultures of neonatal rat ventricular myocytes have become a widely used model to examine a variety of functional, physiological and biochemical cardiac properties. In the adult rat, connexin43 (Cx43) is the major gap junction protein present in the working myocardium. In situ hybridization studies on developing rats, however, showed that Cx40 mRNA displays a dynamic and heterogeneous pattern of expression in the ventricular myocardium around birth. The present studies were performed to examine the expression pattern of the Cx40 protein in neonatal rat heart, and to examine the connexins present in cultures of ventricular myocytes obtained from those hearts.
Cryosections were made of hearts of 1-day-old Wistar rats. Cultures of ventricular myocytes obtained from these hearts by enzymatic dissociation were seeded at various densities (to obtain > 75, approximately 50%, and < 25% confluency) and cultured for 24, 48 or 96 h. Cx40 and Cx43 were detected by immunofluorescence and immunoblotting.
Immunohistochemical stainings confirmed that gap junctions in the atrium and His-Purkinje system were composed of at least Cx43 and Cx40. From the subendocardium towards the subepicardium Cx40 expression gradually decreased, resulting in the sole expression of Cx43 in the subepicardial part of the ventricular wall. In ventricular myocytes cultured at high density (> 75% confluency) Cx43 and Cx40 immunoreactivity could be detected. In contrast to Cx43 immunolabeling which showed a homogeneous distribution pattern, Cx40 staining was heterogeneous, i.e. in some clusters of cells abundant labeling was present whereas in others no Cx40 staining could be detected. The pattern of Cx43 immunoreactivity was not altered by the culture density. In contrast, in isolated ventricular myocytes cultured at low density (< 25% confluency) the relative number of cell-cell interfaces that were Cx40-immunopositive decreased as compared to high density cultures (35 vs. 70%). Western blots did not reveal significant differences in the level of Cx40 and Cx43 expression at different culture densities.
These results show that cultured ventricular myocytes retained typical features of the native neonatal rat ventricular myocardium with regard to their composition of gap junctions. This implicates that these cultures may serve as a good model for studying short-term and long-term regulation of cardiac gap junction channel expression and function.
新生大鼠心室肌细胞原代培养已成为一种广泛应用的模型，用于研究各种心脏功能、生理和生化特性。在成年大鼠中，连接蛋白43（Cx43）是工作心肌中主要的缝隙连接蛋白。然而，对发育中大鼠的原位杂交研究表明，Cx40 mRNA在出生前后的心室心肌中呈现动态且异质性的表达模式。本研究旨在检测新生大鼠心脏中Cx40蛋白的表达模式，并检测从这些心脏获得的心室肌细胞培养物中存在的连接蛋白。
制备1日龄Wistar大鼠心脏的冰冻切片。通过酶解从这些心脏获得的心室肌细胞培养物以不同密度接种（以获得>75%、约50%和<25%的汇合度），并培养24、48或96小时。通过免疫荧光和免疫印迹检测Cx40和Cx43。
免疫组织化学染色证实，心房和希氏-浦肯野系统中的缝隙连接至少由Cx43和Cx40组成。从心内膜下层到心外膜下层，Cx40表达逐渐降低，导致心室壁心外膜部分仅表达Cx43。在高密度（>75%汇合度）培养的心室肌细胞中可检测到Cx43和Cx40免疫反应性。与呈现均匀分布模式的Cx43免疫标记不同，Cx40染色是异质性的，即，在一些细胞簇中有丰富的标记，而在其他细胞簇中未检测到Cx40染色。Cx43免疫反应性模式不受培养密度的影响。相反，在低密度（<25%汇合度）培养的分离心室肌细胞中，与高密度培养相比，Cx40免疫阳性的细胞-细胞界面相对数量减少（35%对70%）。蛋白质免疫印迹未显示不同培养密度下Cx40和Cx43表达水平有显著差异。
这些结果表明，培养的心室肌细胞在缝隙连接组成方面保留了新生大鼠心室心肌的典型特征。这意味着这些培养物可作为研究心脏缝隙连接通道表达和功能的短期和长期调节的良好模型。
