Reinecke H, MacDonald G H, Hauschka S D, Murry C E
Department of Pathology, University of Washington, Seattle, Washington 98195, USA.
J Cell Biol. 2000 May 1;149(3):731-40. doi: 10.1083/jcb.149.3.731.
Skeletal myoblasts form grafts of mature muscle in injured hearts, and these grafts contract when exogenously stimulated. It is not known, however, whether cardiac muscle can form electromechanical junctions with skeletal muscle and induce its synchronous contraction. Here, we report that undifferentiated rat skeletal myoblasts expressed N-cadherin and connexin43, major adhesion and gap junction proteins of the intercalated disk, yet both proteins were markedly downregulated after differentiation into myo-tubes. Similarly, differentiated skeletal muscle grafts in injured hearts had no detectable N-cadherin or connexin43; hence, electromechanical coupling did not occur after in vivo grafting. In contrast, when neonatal or adult cardiomyocytes were cocultured with skeletal muscle, approximately 10% of the skeletal myotubes contracted in synchrony with adjacent cardiomyocytes. Isoproterenol increased myotube contraction rates by 25% in coculture without affecting myotubes in monoculture, indicating the cardiomyocytes were the pacemakers. The gap junction inhibitor heptanol aborted myotube contractions but left spontaneous contractions of individual cardiomyocytes intact, suggesting myotubes were activated via gap junctions. Confocal microscopy revealed the expression of cadherin and connexin43 at junctions between myotubes and neonatal or adult cardiomyocytes in vitro. After microinjection, myotubes transferred dye to neonatal cardiomyocytes via gap junctions. Calcium imaging revealed synchronous calcium transients in cardiomyocytes and myotubes. Thus, cardiomyocytes can form electromechanical junctions with some skeletal myotubes in coculture and induce their synchronous contraction via gap junctions. Although the mechanism remains to be determined, if similar junctions could be induced in vivo, they might be sufficient to make skeletal muscle grafts beat synchronously with host myocardium.
骨骼肌成肌细胞在受损心脏中形成成熟肌肉移植物,这些移植物在受到外源刺激时会收缩。然而,尚不清楚心肌是否能与骨骼肌形成电机械连接并诱导其同步收缩。在此,我们报告未分化的大鼠骨骼肌成肌细胞表达N-钙黏蛋白和连接蛋白43,这两种蛋白是闰盘的主要黏附蛋白和缝隙连接蛋白,但在分化为肌管后,这两种蛋白均显著下调。同样,受损心脏中的分化骨骼肌移植物未检测到N-钙黏蛋白或连接蛋白43;因此,体内移植后未发生电机械偶联。相反,当新生或成年心肌细胞与骨骼肌共培养时,约10%的骨骼肌肌管与相邻心肌细胞同步收缩。异丙肾上腺素使共培养中的肌管收缩率提高了25%,而对单培养中的肌管没有影响,这表明心肌细胞是起搏器。缝隙连接抑制剂庚醇使肌管收缩停止,但单个心肌细胞的自发收缩不受影响,这表明肌管是通过缝隙连接被激活的。共聚焦显微镜显示体外肌管与新生或成年心肌细胞之间的连接处有钙黏蛋白和连接蛋白43的表达。显微注射后,肌管通过缝隙连接将染料转移至新生心肌细胞。钙成像显示心肌细胞和肌管中存在同步钙瞬变。因此,心肌细胞可在共培养中与一些骨骼肌肌管形成电机械连接,并通过缝隙连接诱导其同步收缩。尽管其机制仍有待确定,但如果能在体内诱导出类似的连接,它们可能足以使骨骼肌移植物与宿主心肌同步跳动。